Novel peptides and combination of peptides for use in immunotherapy against lung cancer, including nsclc and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 16/305,686, filed Nov. 29, 2018, which is aNational Stage entry of International Application No. PCT/EP2016/059053,filed Apr. 22, 2016, which claims priority to Great Britain PatentApplication No. 1507030.3, filed Apr. 24, 2015, and U.S. ProvisionalPatent Application 62/152,258, filed Apr. 24, 2015.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-044006_ST25.txt” createdon 29 Jun. 2020, and 29,080 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

BACKGROUND Field

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I and HLA class II molecules ofhuman tumor cells that can be used in vaccine compositions for elicitinganti-tumor immune responses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

Description of Related Art

Lung cancer accounts for the most cancer-related deaths in both men andwomen. Worldwide, lung cancer is the most common cancer in terms of bothincidence and mortality. In 2012, there were more than 1.8 million newcases (13% of total cancer incidence), and 1.6 million deaths (20% oftotal cancer mortality) due to lung cancer. Lung cancer is the leadingcause of cancer death in men in 87 countries and in women in 26countries. More than one third of all newly diagnosed cases occurred inChina. The highest rates are in North America, Europe, and East Asia(World Cancer Report, 2014).

Since 1987, more women have died each year from lung cancer than frombreast cancer. Death rates have continued to decline significantly inmen from 1991-2003 by about 1.9% per year. Female lung cancer deathrates are approaching a plateau after continuously increasing forseveral decades. These trends in lung cancer mortality reflect thedecrease in smoking rates over the past 30 years.

An estimated 230,000 new cases of lung cancer and 160,000 deaths due tolung cancer are expected in 2013 in the USA according to the nationalcancer institute (NCI).

Historically, small cell lung carcinoma has been distinguished fromnon-small cell lung carcinoma (NSCLC), which includes the histologicaltypes of adenocarcinoma, squamous cell carcinoma, and large cellcarcinoma. However, in the past decade, the distinction betweenadenocarcinoma and squamous cell carcinoma has been increasinglyrecognized because of major differences in genetics and also inresponses to specific therapies. Therefore, lung cancers areincreasingly classified according to molecular subtypes, predicated onparticular genetic alterations that drive and maintain lungtumorigenesis (Travis et al., 2013).

Prognosis is generally poor. Of all people with lung cancer, 10-15%survive for five years after diagnosis. Poor survival of lung cancerpatients is due, at least in part, to 80% of patients being diagnosedwith metastatic disease and more than half of patients having distantmetastases (SEER Stat facts, 2014). At presentation, 30-40% of cases ofNSCLC are stage IV, and 60% of SCLC are stage IV.

The 1-year relative survival for lung cancer has slightly increased from35% in 1975-1979 to 44% in 2010, largely due to improvements in surgicaltechniques and combined therapies. However, the 5-year survival rate forall stages combined is only 17%. The survival rate is 54% for casesdetected when the disease is still localized; however, only 16% of lungcancers are diagnosed at this early stage (SEER Stat facts, 2014).

Treatment options are determined by the type (small cell or non-smallcell) and stage of cancer and include surgery, radiation therapy,chemotherapy, and targeted biological therapies such as bevacizumab(AVASTIN®) and erlotinib (TARCEVA®). For localized cancers, surgery isusually the treatment of choice. Recent studies indicate that survivalwith early-stage, non-small cell lung cancer is improved by chemotherapyfollowing surgery. Because the disease has usually spread by the time itis discovered, radiation therapy and chemotherapy are often used,sometimes in combination with surgery. Chemotherapy alone or combinedwith radiation is the usual treatment of choice for small cell lungcancer; on this regimen, a large percentage of patients experienceremission, which is long lasting in some cases surgery (S3-LeitlinieLungenkarzinom, 2011).

Advanced lung cancer has also been resistant to traditionalchemotherapy. However, recent advances have led to exciting progress intherapies that are dependent on histology and genetics. The level ofscrutiny is exemplified by trials of adjuvant chemotherapy designed todifferentiate not only between mutations in codons 12 and 13 of KRAS,but also between different amino acid substitutions as determined byparticular mutations at codon 12 (Shepherd et al., 2013).

To expand the therapeutic options for NSCLC, different immunotherapeuticapproaches have been studied or are still under investigation. Whilevaccination with L-BLP25 or MAGEA3 failed to demonstrate avaccine-mediated survival advantage in NSCLC patients, an allogeneiccell line-derived vaccine showed promising results in clinical studies.Additionally, further vaccination trials targeting gangliosides, theepidermal growth factor receptor and several other antigens arecurrently ongoing. An alternative strategy to enhance the patient'santi-tumor T cell response consists of blocking inhibitory T cellreceptors or their ligands with specific antibodies. The therapeuticpotential of several of these antibodies, including ipilimumab,nivolumab, pembrolizumab, MPDL3280A and MEDI-4736, in NSCLC is currentlyevaluated in clinical trials (Reinmuth et al., 2015).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and lung cancer, including NSCLC inparticular. There is also a need to identify factors representingbiomarkers for cancer in general and lung cancer in particular, leadingto better diagnosis of cancer, assessment of prognosis, and predictionof treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor-associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anti-cancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as beta-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific norover-expressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T cell-based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor-specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen-presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized by CD4-positivehelper T cells bearing the appropriate TCR. It is well known that theTCR, the peptide and the MHC are thereby present in a stoichiometricamount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T cell epitopes derived fromtumor-associated antigens (TAA) is of great importance for thedevelopment of pharmaceutical products for triggering anti-tumor immuneresponses (Gnjatic et al., 2003). At the tumor site, T helper cells,support a cytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara etal., 2006) and attract effector cells, e.g. CTLs, natural killer (NK)cells, macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T helper cell epitopes that trigger a T helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFN-gamma) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC class I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor-associated antigen, leads to an in vitro orin vivo T cell response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T cell response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 110 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 110, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 162, preferably of SEQ ID NO: 1to SEQ ID NO: 110 or a variant thereof, which is at least 77%,preferably at least 88%, homologous (preferably at least 77% or at least88% identical) to SEQ ID NO: 1 to SEQ ID NO: 110, wherein said peptideor variant thereof has an overall length of between 8 and 100,preferably between 8 and 30, and most preferred of between 8 and 20amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D show the over-presentation of various peptides in normaltissues and NSCLC samples; FIGS. 1E-1G show all cell lines, normaltissues and cancers tissues where the exemplary peptides (FVFSFPVSV, SEQID NO: 4 (A*02) and YYTKGFALLNF, SEQ ID NO: 29 (A*24)) has beendetected. FIG. 1A—Gene: SLC6A14, Peptide: FLIPYAIML (A*02; SEQ IDNO.:2)—Tissues from left to right: 1 adipose tissues, 3 adrenal glands,5 arteries, 3 bone marrows, 8 brains, 3 breasts, 13 colons, 1 duodenum,7 esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 4 leukocyte samples,21 livers, 1 lymph node, 1 ovary, 7 pancreas, 2 peripheral nerves, 1peritoneum, 1 pituitary gland, 1 placenta, 3 pleuras, 6 recti, 2salivary glands, 3 skeletal muscles, 3 skins, 2 small intestines, 4spleens, 7 stomachs, 3 testis, 2 thymi, 3 thyroid glands, 1 ureter, 2uteri, 2 veins, 46 lungs, 91 NSCLC. The peptide was also found onpancreatic cancer, gastric cancer, colorectal cancer, esophageal cancer(not shown). FIG. 1B—Gene: COL6A3, Peptide: FLFDGSANL (A*02; SEQ IDNO.:13)—Tissues from left to right: 1 adipose tissues, 3 adrenal glands,5 arteries, 3 bone marrows, 8 brains, 3 breasts, 13 colons, 1 duodenum,7 esophagi, 2 gallbladders, 5 hearts, 16 kidneys, 4 leukocyte samples,21 livers, 1 lymph node, 1 ovary, 7 pancreas, 2 peripheral nerves, 1peritoneum, 1 pituitary gland, 1 placenta, 3 pleuras, 6 recti, 2salivary glands, 3 skeletal muscles, 3 skins, 2 small intestines, 4spleens, 7 stomachs, 3 testis, 2 thymi, 3 thyroid glands, 1 ureter, 2uteri, 2 veins, 46 lungs, 91 NSCLC. The peptide was also found onprostate cancer, breast cancer, colorectal cancer, hepatic cancer,melanoma, ovarian cancer, esophageal cancer, pancreatic cancer, gastriccancer (not shown). FIG. 1C—Gene: CCL18, Peptide: VYTSWQIPQKF (A*24; SEQID NO.:23)—Tissues from left to right: 2 adrenal glands, 1 artery, 4brains, 1 breast, 5 colons, 1 heart, 13 kidneys, 9 livers, 3 pancreas, 1pituitary gland, 2 recti, 3 skins, 1 spleen, 12 stomachs, 1 thymus, 2uteri, 9 lungs, 80 NSCLC. The peptide was also found on prostate cancer,gastric cancer (not shown). FIG. 1D—Gene: CENPN, Peptide: RYLDSLKAIVF(A*24; SEQ ID NO.:28)—Tissues from left to right: 2 adrenal glands, 1artery, 4 brains, 1 breast, 5 colons, 1 heart, 13 kidneys, 9 livers, 3pancreas, 1 pituitary gland, 2 recti, 3 skins, 1 spleen, 12 stomachs, 1thymus, 2 uteri, 9 lungs, 80 NSCLC. The peptide was also found onhepatic cancer, gastric cancer, RCC (not shown). FIG. 1E—Gene: DUSP4,Peptide: FVFSFPVSV (A*02; SEQ ID NO.:4)—Tissues from left to right: 5pancreatic cell lines, 3 skins, 15 normal tissues (2 esophagi, 7 lungs,3 spleens, 3 stomachs), 126 cancer tissues (1 brain cancer, 2 breastcancers, 5 colon cancers, 5 esophageal cancers, 2 gallbladder cancers, 8kidney cancers, 5 liver cancers, 58 lung cancers, 11 ovarian cancers, 9pancreatic cancers, 2 prostate cancers, 1 rectal cancer, 4 skin cancers,12 stomach cancers, 1 testis cancer). The set of normal tissues was thesame as in A-B, but tissues without detection are not shown.

FIG. 1F—Gene: PLOD2, Peptide: YYTKGFALLNF (A*24; SEQ ID NO.:29)—Tissuesfrom left to right: 30 cancer tissues (1 brain cancer, 3 kidney cancers,2 liver cancers, 22 lung cancers, 2 stomach cancers). The set of normaltissues was the same as in C-D, but tissues without detection are notshown. FIG. 1G show the over-presentation of an A*24 peptide in normaltissues and NSCLC samples. Gene: LAMP3, Peptide: RFMDGHITF (A*24; SEQ IDNO.:25)—Tissues from left to right: 2 adrenal glands, 1 artery, 4brains, 1 breast, 5 colons, 1 heart, 13 kidneys, 9 livers, 3 pancreas, 1pituitary gland, 2 recti, 3 skins, 1 spleen, 12 stomachs, 1 thymus, 2uteri, 9 lungs, 80 NSCLC. The peptide was also found on prostate cancer,gastric cancer (not shown).

FIGS. 2A-2D show exemplary expression profiles (relative expressioncompared to normal kidney) of source genes of the present invention thatare highly over-expressed or exclusively expressed in lung cancer in apanel of normal tissues and 38 lung cancer samples. Tissues from left toright: adrenal gland, artery, bone marrow, brain (whole), breast, colon,esophagus, heart, kidney (triplicate), leukocytes, liver, lung, lymphnode, ovary, pancreas, placenta, prostate, salivary gland, skeletalmuscle, skin, small intestine, spleen, stomach, testis, thymus, thyroidgland, urinary bladder, uterine cervix, uterus, vein, 1 normal (healthy)lung sample, 38 NSCLC samples. FIG. 2A: SMC4; FIG. 2B: LAMB3; FIG. 2C:MMP12; and FIG. 2D: LAMP3.

FIGS. 3A-3E show exemplary immunogenicity data: flow cytometry resultsafter peptide-specific multimer staining. FIG. 3A: SLC1A4-001 (SEQ IDNo. 12); FIG. 3B: IGF2BP3-001 (SEQ ID No. 120); FIG. 3C: LAMC2-001 (SEQID No. 121); FIG. 3D: COL6A3-008 (SEQ ID No. 13); and FIG. 3E: LAMP3-001(SEQ ID No. 25).

FIG. 4 shows the results of antigen stimulated CD4+ T-cellproliferation: The figure shows the number of positive donors for eachpeptide.

FIG. 5 shows exemplary vaccine-induced CD4 T-cell response to CEA-006 inclass II ICS assay. Following in vitro sensitization PBMCs of patient36-031 were analyzed for CD4 T-cell responses to CEA-006 (upper panel)and mock (lower panel) at time point pool V8/EOS. Cells were stimulatedwith corresponding peptides and stained with viability, anti-CD3,anti-CD8, anti-CD4 and effector markers (from right to left: CD154,TNF-alpha, IFN-gamma, IL-2, IL-10), respectively. Viable CD4 T-cellswere analyzed for the proportion of cells positive for one or moreeffector molecules.

FIG. 6 shows the immunogenicity of control class II peptides: Thediagram shows the immune response rate to 5 class II peptides detectedin 16 patients for IMA950 peptides and in 71 patients for IMA910peptides using ICS.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table4 bind to HLA-A*02. All peptides in Table 2 bind to HLA-A*24. Allpeptides in Table 3 and Table 5 bind to HLA-DR. The peptides in Table 4and Table 5 have been disclosed before in large listings as results ofhigh-throughput screenings with high error rates or calculated usingalgorithms, but have not been associated with cancer at all before. Thepeptides in Table 6, Table 7, and Table 8 are additional peptides thatmay be useful in combination with the other peptides of the invention.The peptides in Table 9 and Table 10 are furthermore useful in thediagnosis and/or treatment of various other malignancies that involve anover-expression or over-presentation of the respective underlyingpolypeptide.

TABLE 1 Peptides according to the present invention SEQ ID Official NoSequence Gene ID(s) Gene Symbol(s)  1 KLLPYIVGV 1293 COL6A3  2 FLIPYAIML11254 SLC6A14  3 FLYDVVKSL 1293 COL6A3  4 FVFSFPVSV 1846 DUSP4  5ALTSTLISV 10457 GPNMB  6 SLQGSIMTV 653509, 729238 SFTPA1, SFTPA2  7NLLQVLEKV 144501 KRT80  8 ALLNILSEV 55236 UBA6  9 ALSGTLSGV 4174 MCM5 10KMAGIGIREA 3866 KRT15 11 YLNVQVKEL 10051 SMC4 12 IVDRTTTVV 6509 SLC1A413 FLFDGSANL 1293 COL6A3 14 LIQDRVAEV 3914 LAMB3 15 ELDRTPPEV 23450SF3B3 16 LIFDLGGGTFDV 3303, 3304, HSPA1A, HSPA1B, 3305, 3306, HSPA1L, HSPA2, 3310, 3311, HSPA6, HSPA7, 3312 HSPA8 17 TLLQEQGTKTV286887, 3852, KRT6C, KRT5, 3853, 3854 KRT6A, KRT6B 18 ILLTEQINL10745, 57157 PHTF1, PHTF2 19 VLTSDSPAL 10457 GPNMB 20 LMTKEISSV 5591PRKDC 21 VLSSGLTAA 1459 CSNK2A2 22 NLINQEIML 5783 PTPN13

TABLE 2 Additional Peptides according to the present invention SEQOfficial ID No Sequence Gene ID(s) Gene Symbol(s) 23 VYTSWQIPQKF101060271, 6362 CCL18 24 NYPKSIHSF 4321 MMP12 25 RFMDGHITF 27074 LAMP326 RYLEKFYGL 4321 MMP12 27 RYPPPVREF 1293 COL6A3 28 RYLDSLKAIVF 55839CENPN 29 YYTKGFALLNF 5352 PLOD2 30 KYLEKYYNL 4312 MMP1 31 SYLDKVRAL3858, 3859, 3860, KRT10, KRT12, KRT13, 3861, 3866, 3868,KRT14, KRT15, KRT16, 3872, 3880 KRT17, KRT19 32 EYQPEMLEKF 1293 COL6A333 TYSEKTTLF 94025 MUC16 34 VFMKDGFFYF 4312 MMP1 35 TYNPEIYVI 3673 ITGA236 YYGNTLVEF 25903 OLFML2B 37 RYLEYFEKI 79573 TTC13 38 VFLNRAKAVFF 10457GPNMB 39 KFLEHTNFEF 1794 DOCK2 40 IYNPSMGVSVL 5818 PVRL1 41 TYIGQGYII60681 FKBP10 42 VYVTIDENNIL 4363 ABCC1 43 RYTLHINTL 247 ALOX15B 44IYNQIAELW 27293 SMPDL3B 45 KFLESKGYEF 9945 GFPT2 46 NYTNGSFGSNF 1655DDX5 47 RYISPDQLADL 2023 ENO1 48 YYYGNTLVEF 25903 OLFML2B 49 QYLFPSFETF3824 KLRD1 50 LYIGWDKHYGF 5685 PSMA4 51 NYLLESPHRF 9842 PLEKHM1 52SYMEVPTYLNF 7805 LAPTM5 53 IYAGQWNDF 81035 COLEC12 54 AYKDKDISFF 58486ZBED5 55 IYPVKYTQTF 64065 PERP 56 RYFPTQALNF 291, 292, 293,SLC25A4, SLC25A5, 83447 SLC25A6, SLC25A31 57 SYSIGIANF 1303 COL12A1 58VYFKPSLTPSGEF 9972 NUP153 59 HYFNTPFQL 160760 PPTC7 60 SYPAKLSFI 4029L1RE1 61 RYGSPINTF 647024 C6orf132 62 AYKPGALTF 84883 AIFM2 63 LYINKANIW55632 G2E3 64 VYPLALYGF 9213 XPR1 65 IYQRWKDLL 219285 SAMD9L 66DYIPQLAKF 2744 GLS 67 IFLDYEAGHLSF 81559 TRIM11 68 RYLFVVDRL 55686 MREG69 TYAALNSKATF 8826 IQGAP1 70 VYHSYLTIF 7226 TRPM2 71 TYLTNHLRL 90874ZNF697 72 YYVDKLFNTI 5922 RASA2 73 RYLHVEGGNF 3516 RBPJ 74 EYLPEFLHTF154664 ABCA13 75 AYPDLNEIYRSF 11262 SP140 76 VYTZIQSRF 8445, 8798DYRK2, DYRK4 77 RYLEAGAAGLRW 23640 HSPBP1 78 IYTRVTYYL 64499, 7177TPSB2, TPSAB1 79 RYGGSFAEL 23135 KDM6B 80 AYLKEVEQL 8087 FXR1 81KYIEAIQWI 81501 DCSTAMP 82 FYQGIVQQF 10426 TUBGCP3 83 EYSDVLAKLAF 27245AHDC1 84 TFDVAPSRLDF 23420, 283820, NOMO1, NOMO2, NOMO3 408050 85PFLQASPHF 84985 FAM83A

TABLE 3 HLA-DR peptides according to the present invention SEQ GeneOfficial ID No Sequence ID(s) Gene Symbol(s) 86 LSADDIRGIQSLYGDPK 4321MMP12 87 EGDIQQFLITGDPKAAYDY 1301 COL11A1 88 NPVSQVEILKNKPLSVG 3694ITGB6 89 KLYIGNLSENAAPS 10643 IGF2BP3 90 DAVQMVITEAQKVDTR 3918 LAMC2 91VARLPIIDLAPVDVGGTD 1290 COL5A2 92 NKPSRLPFLDIAPLDIGGAD 1278 COL1A2 93SRPQAPITGYRIVYSPSV 2335 FN1

TABLE 4 Additional peptides according to the presentinvention with no prior known cancer association SEQ Official ID NoSequence Gene ID(s) Gene Symbol(s)  94 ILVDWLVQV 9133 CCNB2  95KIIGIMEEV 2956 MSH6  96 AMGIAPPKV 9129 PRPF3  97 TLFPVRLLV 79888 LPCAT1 98 VLYPHEPTAV 29980, 5523 DONSON, PPP2R3A  99 ALFQRPPLI 1736 DKC1 100KIVDFSYSV 701 BUB1B 101 LLLEILHEI 30001 ERO1L 102 SLLSELQHA 115362 GBPS103 KLLSDPNYGV 79188 TMEM43 104 SLVAVELEKV 25839 COG4 105 IVAESLQQV 6772STAT1 106 SILEHQIQV 4173 MCM4 107 ALSERAVAV 10213 PSMD14 108 TLLDFINAV55236 UBA6 109 NLIEVNEEV 221960, 51622 CCZ1B, CCZ1

TABLE 5 Additional HLA-DR peptides according to thepresent invention with no prior known cancer association SEQ GeneOfficial ID No Sequence ID(s) Gene Symbol(s) 110 IQLIVQDKESVFSPR 27074LAMP3

TABLE 6 Other peptides useful for e.g. personalized cancer therapies SEQOfficial ID No Sequence Gene ID(s) Gene Symbol(s) 111 SLYKGLLSV 25788RAD54B 112 VLAPLFVYL 2535, 8321, FZD2, FZD1, FZD7 8324 113 FLLDGSANV1293 COL6A3 114 AMSSKFFLV 7474 WNT5A 115 YVYQNNIYL 2191 FAP 116KIQEMQHFL 4321 MMP12 117 ILIDWLVQV 891 CCNB1 118 SLHFLILYV 487, 488ATP2A1, ATP2A2 119 IVDDITYNV 2335 FN1 120 KIQEILTQV 10643 IGF2BP3 121RLLDSVSRL 3918 LAMC2 122 KLSWDLIYL 51148 CERCAM 123 GLTDNIHLV 25878MXRA5 124 NLLDLDYEL 1293 COL6A3 125 RLDDLKMTV 3918 LAMC2 126 KLLTEVHAA101 ADAM8 127 ILFPDIIARA 64110 MAGEF1 128 TLSSIKVEV 25878 MXRA5 129GLIEIISNA 23020 SNRNP200 130 KILEDWGV 22974 TPX2 131 ALVQDLAKA 891 CCNB1132 ALFVRLLALA 7045 TGFBI 133 RLASYLDKV 3857, 3858, KRT9, KRT10, 3859, 3860, KRT12, KRT13, 3861, 3866, KRT14, KRT15, 3868, 3872,KRT16, KRT17, 3880 KRT19 134 TLWYRAPEV 1019, 1021 CDK4, CDK6 135AIDGNNHEV 9945 GFPT2 136 ALVDHTPYL 1462 VCAN 137 FLVDGSWSV 1303 COL12A1138 ALNEEAGRLLL 27338 UBE2S 139 SLIEDLILL 64754 SMYD3 140 TLYPHTSQV 1462VCAN 141 NLIEKSIYL 667 DST 142 VLLPVEVATHYL 10568 SLC34A2 143 AIVDKVPSV22820 COPG1 144 KIFDEILVNA 7153, 7155 TOP2A, TOP2B 145 AMTQLLAGV 3371TNC 146 FQYDHEAFL 57333, 5954 RCN3, RCN1 147 VLFPNLKTV 646 BNC1 148ALFGALFLA 5360 PLTP 149 KLVEFDFLGA 10460 TACC3 150 GVLENIFGV 399909PCNXL3 151 AVVEFLTSV 29102 DROSHA 152 ILQDRLNQV 990 CDC6 153 ALYDSVILL1734 DIO2 154 ILFEINPKL 154664 ABCA13 155 ALDENLHQL 154664 ABCA13 156TVAEVIQSV 55083 KIF26B 157 KLFGEKTYL 6317 SERPINB3 158 KLDETNNTL 667 DST

TABLE 7 Other peptides useful for e.g. personalized cancer therapies SEQGene Official ID No Sequence ID(s) Gene Symbol(s) 159 TYKYVDINTF 4321MMP12 160 SYLQAANAL 1293 COL6A3 161 LYQILQGIVF 983 CDK1

TABLE 8 HLA-DR peptides useful for e.g. personalized cancer therapiesSEQ Gene Official ID No Sequence ID(s) Gene Symbol(s) 162TNGVIHWDKLLYPADT 10631 POSTN

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, brain cancer, breastcancer, colorectal cancer, esophageal cancer, kidney cancer, livercancer, ovarian cancer, pancreatic cancer, prostate cancer, gastriccancer, melanoma, merkel cell carcinoma, leukemia (AML, CLL),non-Hodgkin lymphoma (NHL), esophageal cancer including cancer of thegastric-esophageal junction (OSCAR), gallbladder cancer andcholangiocarcinoma (GBC_CCC), urinary bladder cancer (UBC), uterinecancer (UEC).

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 110. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 14 (see Table 1) and SEQ ID NO: 23 to SEQ IDNO: 47 (see Table 2), and their uses in the immunotherapy of lung cancer(including NSCLC), brain cancer, breast cancer, colorectal cancer,esophageal cancer, kidney cancer, liver cancer, ovarian cancer,pancreatic cancer, prostate cancer, gastric cancer, melanoma, merkelcell carcinoma, leukemia (AML, CLL), non-Hodgkin lymphoma (NHL),esophageal cancer including cancer of the gastric-esophageal junction(OSCAR), gallbladder cancer and cholangiocarcinoma (GBC_CCC), urinarybladder cancer (UBC), uterine cancer (UEC), and preferably lung cancer,including NSCLC.

As shown in the following Table 9, 9-2, and Table 10 and 10-2, many ofthe peptides according to the present invention are also found on othertumor types and can, thus, also be used in the immunotherapy of otherindications. Also refer to FIG. 1 and Example 1.

TABLE 9HLA-A*02 peptides according to the present invention and their specific uses in otherproliferative diseases, especially in other cancerous diseases. The table shows forselected peptides on which additional tumour types they were found and either over-presented on more than 5% of the measured tumour samples, or presented on more than 5% ofthe measured tumour samples with a ratio of geometric means tumour vs normal tissuesbeing larger than 3. SEQ ID No. Sequence Other relevant organs/diseases  1 KLLPYIVGV Pancreas, Breast   2 FLIPYAIMLStomach, Colon, Rectum, Pancreas   3 FLYDVVKSL Pancreas, Breast   4FVFSFPVSV Stomach, Pancreas, Breast, Melanoma, Ovary   5 ALTSTLISVBreast, Melanoma, Esophagus   7 NLLQVLEKVKidney, Colon, Rectum, Liver, Breast   8 ALLNILSEVBrain, Liver, Prostate, Ovary   9 ALSGTLSGVBrain, Liver, Leukocytes, Melanoma, Ovary, Esophagus  10 KMAGIGIREAProstate, Ovary  11 YLNVQVKEL Colon, Rectum, Liver  13 FLFDGSANLColon, Rectum, Pancreas, Breast, Esophagus  14 LIQDRVAEV Kidney  15ELDRTPPEV Kidney, Brain, Liver, Leukocytes  16 LIFDLGGGTFDVBrain, Liver, Prostate, Breast, Melanoma, Ovary  18 ILLTEQINLKidney, Stomach, Liver, Pancreas, Prostate, Breast, Ovary, Esophagus  19VLTSDSPAL Liver, Melanoma, Esophagus  20 LMTKEISSVBrain, Liver, Melanoma  21 VLSSGLTAA Liver, Esophagus  94 ILVDWLVQVKidney, Brain, Stomach, Colon, Rectum, Liver, Melanoma, Ovary  95KIIGIMEEV Kidney, MCC, Esophagus  96 AMGIAPPKVColon, Rectum, Liver, Pancreas, Leukocytes  97 TLFPVRLLVKidney, Leukocytes  98 VLYPHEPTAVKidney, Brain, Colon, Rectum, Liver, MCC, Melanoma, Ovary  99 ALFQRPPLIColon, Rectum, Liver, Ovary 100 KIVDFSYSVBrain, Colon, Rectum, MCC, Ovary 101 LLLEILHEIKidney, Liver, Pancreas, Breast, Ovary 102 SLLSELQHAKidney, Breast, Ovary, Esophagus 103 KLLSDPNYGV Brain, Pancreas 104SLVAVELEKV Brain, Liver, Pancreas, MCC, Ovary, Esophagus 105 IVAESLQQVKidney, Stomach, Pancreas, Breast, Ovary, Esophagus 106 SILEHQIQV Kidney111 SLYKGLLSV Kidney, Brain, Colon, Rectum, Liver, Ovary 112 VLAPLFVYLKidney, Pancreas, Breast, Melanoma 113 FLLDGSANVStomach, Colon, Rectum, Liver, Pancreas, Breast, Ovary, Esophagus 114AMSSKFFLVBrain, Stomach, Colon, Rectum, Liver, Pancreas, Prostate, Ovary,Esophagus 115 YVYQNNIYLStomach, Colon, Rectum, Liver, Pancreas, Breast, Melanoma,Ovary, Esophagus 116 KIQEMQHFL Colon, Rectum 117 ILIDWLVQVKidney, Brain, Stomach, Colon, Rectum, Liver, Pancreas, Melanoma, Ovary118 SLHFLILYV Kidney, Brain, Colon, Rectum, Liver, Melanoma, Ovary 119IVDDITYNV Liver, Pancreas, Breast, Esophagus 120 KIQEILTQVKidney, Brain, Stomach, Colon, Rectum, Liver, Pancreas,Leukocytes, Ovary, Esophagus 121 RLLDSVSRLKidney, Colon, Rectum, Liver, Pancreas, Ovary 122 KLSWDLIYLKidney, Colon, Rectum 123 GLTDNIHLVKidney, Colon, Rectum, Pancreas, Ovary, Esophagus 124 NLLDLDYELStomach, Colon, Rectum, Pancreas, Breast, Ovary, Esophagus 125 RLDDLKMTVColon, Rectum, Pancreas, Ovary, Esophagus 126 KLLTEVHAAKidney, Stomach, Colon, Rectum, Liver, Pancreas, Breast, Ovary 127ILFPDIIARA Kidney, Brain, Leukocytes, Esophagus 128 TLSSIKVEVKidney, Stomach, Colon, Rectum, Pancreas, Prostate, Breast,Melanoma, Ovary 129 GLIEIISNABrain, Colon, Rectum, Liver, Ovary, Esophagus 130 KILEDVVGVKidney, Stomach, Colon, Rectum, Melanoma, Ovary, Esophagus 131 ALVQDLAKAKidney, Stomach, Colon, Rectum, Liver, Pancreas, Ovary 132 ALFVRLLALAKidney, Brain, Stomach, Colon, Rectum, Liver, Pancreas,Melanoma, Ovary, Esophagus 133 RLASYLDKV Pancreas, Breast, Esophagus 134TLWYRAPEV Stomach, Melanoma, Ovary 135 AIDGNNHEV Brain, Liver, Pancreas136 ALVDHTPYL Kidney, Liver, Pancreas 137 FLVDGSWSVStomach, Colon, Rectum, Pancreas, Ovary, Esophagus 138 ALNEEAGRLLLKidney, Brain, Stomach, Colon, Rectum, Liver, Pancreas, MCC,Melanoma, Ovary, Esophagus 139 SLIEDLILLKidney, Brain, Colon, Rectum, Liver, Pancreas, Prostate,Melanoma, Ovary, Esophagus 141 NLIEKSIYL Esophagus 142 VLLPVEVATHYLOvary 143 AIVDKVPSV Kidney, Liver, Pancreas, Prostate, Ovary, Esophagus144 KIFDEILVNA Stomach, Colon, Rectum, Melanoma, Ovary, Esophagus 145AMTQLLAGV Brain, Colon, Rectum, Breast, Esophagus 146 FQYDHEAFLKidney, Stomach, Colon, Rectum, Pancreas, Melanoma, Esophagus 147VLFPNLKTV Kidney 148 ALFGALFLA Melanoma, Ovary 149 KLVEFDFLGABrain, Stomach, Colon, Rectum, Liver, MCC, Ovary, Esophagus 150GVLENIFGV Kidney, Brain, Liver, Ovary, Esophagus 151 AWEFLTSVBrain, MCC, Esophagus 152 ILQDRLNQV Colon, Rectum, Liver, Ovary 153ALYDSVILL Stomach, Prostate, Esophagus 155 ALDENLHQL Esophagus 156TVAEVIQSV Pancreas, Breast, Esophagus 157 KLFGEKTYL Esophagus

TABLE 9-2HLA-A*02 peptides according to the present invention and their specificuses in other proliferative diseases, especially in other cancerous diseases(amendment of Table 9). The table shows, like Table 9, for selected peptides on whichadditional tumor types they were found showing over-presentation (including specificpresentation) on more than 5% of the measured tumor samples, or presentation onmore than 5% of the measured tumor samples with a ratio of geometric means tumorvs normal tissues being larger than 3. Over-presentation is defined as higherpresentation on the tumor sample as compared to the normal sample with highestpresentation. Normal tissues against which over-presentation was tested were:adipose tissue, adrenal gland, blood cells, blood vessel, bone marrow, brain,cartilage, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lung,lymph node, nerve, pancreas, parathyroid gland, peritoneum, pituitary, pleura,salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus,thyroid gland, trachea, ureter, and urinary bladder. SEQ ID No. SequenceAdditional Entities   1 KLLPYIVGVSCLC, CRC, Melanoma, Esophageal Cancer, GallbladderCancer, Bile Duct Cancer   2 FLIPYAIMLSCLC, PC, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer  3 FLYDWKSL SCLC, CRC, Gallbladder Cancer, Bile Duct Cancer   4FVFSFPVSV SCLC, Esophageal Cancer, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer, NHL   7 NLLQVLEKVSCLC, OC, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer   8 ALLNILSEVSCLC, BRCA, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer  9 ALSGTLSGV CLL, BRCA, Urinary bladder cancer, Uterine Cancer,AML, NHL  10 KMAGIGIREA Urinary bladder cancer  11 YLNVQVKELSCLC, Melanoma, Esophageal Cancer, Urinary bladdercancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML  13FLFDGSANL SCLC, GC, Melanoma, OC, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer  14 LIQDRVAEVEsophageal Cancer, Urinary bladder cancer  15 ELDRTPPEVSCLC, PC, CLL, Esophageal Cancer, Urinary bladdercancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL 17 TLLQEQGTKTV SCLC, Esophageal Cancer, Urinary bladder cancer  18ILLTEQINL SCLC, Melanoma, Gallbladder Cancer, Bile Duct Cancer  20LMTKEISSV OC, Esophageal Cancer, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer  21 VLSSGLTAACRC, BRCA, Urinary bladder cancer, Uterine Cancer  22 NLINQEIMLBRCA, Melanoma, Urinary bladder cancer  94 ILVDWLVQVSCLC, BRCA, Esophageal Cancer, Urinary bladdercancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, AML, NHL 95 KIIGIMEEV SCLC, Brain Cancer, GC, CRC, Melanoma, AML, NHL  96AMGIAPPKV SCLC, BRCA, Melanoma, NHL  98 VLYPHEPTAVSCLC, BRCA, Esophageal Cancer, Urinary bladder cancer, NHL  99 ALFQRPPLISCLC, CLL, Esophageal Cancer, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer, NHL 100 KIVDFSYSVSCLC, BRCA, Melanoma, Urinary bladder cancer, Uterine Cancer 101LLLEILHEI SCLC, CLL, Urinary bladder cancer 102 SLLSELQHASCLC, Melanoma, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer, NHL 103 KLLSDPNYGVBRCA, Melanoma, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer 105 IVAESLQQV SCLC, Melanoma, NHL 106 SILEHQIQVCRC, BRCA, Esophageal Cancer, Urinary bladder cancer,Uterine Cancer, AML, NHL 107 ALSERAVAV Esophageal Cancer, Uterine Cancer108 TLLDFINAV NHL 109 NLIEVNEEV CLL, AML SCLC = small cell lung cancer,RCC = kidney cancer, CRC = colon or rectum cancer, GC = stomach cancer,HCC = liver cancer, PC = pancreatic cancer, PrC = prostate cancer, BRCA= breast cancer, MCC = Merkel cell carcinoma, OC = ovarian cancer, NHL= non-Hodgkin lymphoma, AML = acute myeloid leukemia, CLL = chroniclymphocytic leukemia.

TABLE 10 HLA-A*24 peptides according to the presentinvention and their specific uses in otherproliferative diseases, especially in othercancerous diseases. The table shows for selectedpeptides on which additional tumor types theywere found and either over-presented on morethan 5% of the measured tumor samples, orpresented on more than 5% of the measuredtumor samples with a ratio of geometric meanstumor vs normal tissues being larger than 3. SEQ ID No. SequenceOther relevant organs/diseases  26 RYLEKFYGL Stomach, Liver  27RYPPPVREF Liver, Prostate  28 RYLDSLKAIVF Kidney, Liver  29 YYTKGFALLNFKidney, Brain, Liver  31 SYLDKVRAL Stomach  33 TYSEKTTLF Stomach  36YYGNTLVEF Brain, Stomach  37 RYLEYFEKI Brain, Liver, Prostate  38VFLNRAKAVFF Liver  39 KFLEHTNFEF Liver  41 TYIGQGYIIBrain, Stomach, Liver, Prostate  42 VYVTIDENNIL Kidney, Stomach  43RYTLHINTL Prostate  44 IYNQIAELW Stomach, Liver  45 KFLESKGYEF Brain  46NYTNGSFGSNF Liver  47 RYISPDQLADL Kidney  49 QYLFPSFETF Stomach  50LYIGWDKHYGF Kidney, Stomach, Liver  51 NYLLESPHRF Liver  52 SYMEVPTYLNFLiver  53 IYAGQWNDF Prostate  54 AYKDKDISFF Kidney, Brain  56 RYFPTQALNFKidney, Stomach, Liver  58 VYFKPSLTPSGEF Stomach, Liver  59 HYFNTPFQLKidney, Brain, Liver, Prostate  60 SYPAKLSFI Liver  61 RYGSPINTFLiver, Prostate  62 AYKPGALTF Liver  63 LYINKANIW Stomach, Liver  66DYIPQLAKF Kidney, Liver  67 IFLDYEAGHLSF Kidney, Stomach, Liver,Prostate  69 TYAALNSKATF Liver  70 VYHSYLTIF Brain, Liver  71 TYLTNHLRLLiver  72 YYVDKLFNTI Liver, Prostate  73 RYLHVEGGNF Brain, Liver  75AYPDLNEIYRSF Liver  76 VYTZIQSRF Liver, Prostate  77 RYLEAGAAGLRWStomach, Liver  78 IYTRVTYYL Stomach, Prostate  79 RYGGSFAELBrain, Liver  80 AYLKEVEQL Brain, Prostate  81 KYIEAIQWI Liver  82FYQGIVQQF Brain, Liver, Prostate  84 TFDVAPSRLDF Liver, Prostate  85PFLQASPHF Stomach 159 TYKYVDINTF Stomach 160 SYLQAANAL Stomach 161LYQILQGIVF Kidney, Stomach, Liver

TABLE 10-2 HLA-A*24 peptides according to the presentinvention and their specific usesin otherproliferative diseases, especially in othercancerous diseases (amendment of Table 10). Thetable shows, like Table 10, for selected peptideson which additional tumor types they were foundshowing over-presentation (including specificpresentation) on more than 5% of the measuredtumor samples, or presentation on more than 5%of the measured tumor samples with a ratio ofgeometric means tumor vs normal tissues beinglarger than 3. Over-presentation is defined as higher presentation on the tumor sample ascompared to the normal sample with highestpresentation. Normal tissues against which over-presentation was tested were: adrenal gland,artery, brain, heart, kidney, large intestine,liver, lung, pancreas, pituitary, skin, spleen, stomach, thymus.  SEQ IDAdditional No. Sequence Entities 27 RYPPPVREF Brain Cancer 32 EYQPEMLEKFBrain Cancer 40 IYNPSMGVSVL Brain Cancer 46 NYTNGSFGSNF Brain Cancer 47RYISPDQLADL HCC 48 YYYGNTLVEF Brain Cancer 57 SYSIGIANF Brain Cancer 61RYGSPINTF GC 67 IFLDYEAGHLSF Brain Cancer 72 YYVDKLFNTI Brain Cancer 76VYTZIQSRF Brain Cancer GC = stomach cancer, HCC = liver cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 7, 14, 15, 18, 94, 95, 97, 98, 101, 102, 105, 106,111, 112, 117, 118, 120, 121, 122, 123, 126, 127, 128, 130, 131, 132,136, 138, 139, 143, 146, 147, 150, 28, 29, 42, 47, 50, 54, 56, 59, 66,67 and 161 for the—in one preferred embodiment combined—treatment ofkidney cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 8, 9, 15, 16, 20, 94, 98, 100, 103, 104, 111, 114,117, 118, 120, 127, 129, 132, 135, 138, 139, 145, 149, 150, 151, 29, 36,37, 41, 45, 54, 59, 70, 73, 79, 80 and 82 for the—in one preferredembodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 4, 18, 94, 105, 113, 114, 115, 117, 120, 124, 126,128, 130, 131, 132, 134, 137, 138, 144, 146, 149, 153, 26, 31, 33, 36,41, 42, 44, 49, 50, 56, 58, 63, 67, 77, 78, 85, 159, 160 and 161 forthe—in one preferred embodiment combined—treatment of gastric cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 7, 11, 13, 94, 96, 98, 99, 100, 111, 113, 114, 115,116, 117, 118, 120, 121, 122, 123, 124, 125, 126, 128, 129, 130, 131,132, 137, 138, 139, 144, 145, 146, 149 and 152 for the—in one preferredembodiment combined—treatment of colorectal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 7, 8, 9, 11, 15, 16, 18, 19, 20, 21, 94, 96, 98, 99,101, 104, 111, 113, 114, 115, 117, 118, 119, 120, 121, 126, 129, 131,132, 135, 136, 138, 139, 143, 149, 150, 152, 26, 27, 28, 29, 37, 38, 39,41, 44, 46, 50, 51, 52, 56, 58, 59, 60, 61, 62, 63, 66, 67, 69, 70, 71,72, 73, 75, 76, 77, 79, 81, 82, 84 and 161 for the—in one preferredembodiment combined—treatment of liver cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 2, 3, 4, 13, 18, 96, 101, 103, 104, 105, 112, 113,114, 115, 117, 119, 120, 121, 123, 124, 125, 126, 128, 131, 132, 133,135, 136, 137, 138, 139, 143, 146 and 156 for the—in one preferredembodiment combined—treatment of pancreatic cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 8, 10, 16, 18, 114, 128, 139, 143, 153, 27, 37, 41,43, 53, 59, 61, 67, 72, 76, 78, 80, 82 and 84 for the—in one preferredembodiment combined—treatment of prostate cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 9, 15, 96, 97, 120 and 127 for the—in one preferredembodiment combined—treatment of leukemia (AML, CLL).

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 3, 4, 5, 7, 13, 16, 18, 101, 102, 105, 112, 113,115, 119, 124, 126, 128, 133, 145 and 156 for the—in one preferredembodiment combined—treatment of breast cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 95, 98, 100, 104, 138, 149 and 151 for the—in onepreferred embodiment combined—treatment of merkel cell carcinoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 5, 9, 16, 19, 20, 94, 98, 112, 115, 117, 118, 128,130, 132, 134, 138, 139, 144, 146 and 148 for the—in one preferredembodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 8, 9, 10, 16, 18, 94, 98, 99, 100, 101, 102, 104,105, 111, 113, 114, 115, 117, 118, 120, 121, 123, 124, 125, 126, 128,129, 130, 131, 132, 134, 137, 138, 139, 142, 143, 144, 148, 149, 150 and152 for the—in one preferred embodiment combined—treatment of ovariancancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 5, 9, 13, 18, 19, 21, 95, 102, 104, 105, 113, 114,115, 119, 120, 123, 124, 125, 127, 129, 130, 132, 133, 137, 138, 139,141, 143, 144, 145, 146, 149, 150, 151, 153, 155, 156 and 157 for the—inone preferred embodiment combined—treatment of esophageal cancer.

Thus, another particularly preferred aspect of the present inventionrelates to the use of at least one peptide according to the presentinvention according to any one of SEQ ID No. 13, 25, 113, 114, 115, 120,121, 128, 159, and 161 for the—preferably combined—treatment of lungcancer (including NSCLC),

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group oflung cancer (including NSCLC), brain cancer, breast cancer, colorectalcancer, esophageal cancer, kidney cancer, liver cancer, ovarian cancer,pancreatic cancer, prostate cancer, gastric cancer, melanoma, merkelcell carcinoma, leukemia (AML, CLL).

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class I or—in an elongatedform, such as a length-variant—MHC class II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 162, preferably of SEQ ID NO: 1 to SEQ ID NO: 110.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 110, preferably containing SEQ IDNo. 1 to SEQ ID No. 14 and SEQ ID No. 23 to SEQ ID No. 47 or a variantamino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody. Preferably, said medicamentis a cellular therapy, a vaccine or a protein derived from a soluble TCRor antibody, e.g. a sTCR comprising an anti-CD3 antibody or partthereof.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are lung cancer (including NSCLC),brain cancer, breast cancer, colorectal cancer, esophageal cancer,kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostatecancer, gastric cancer, melanoma, merkel cell carcinoma, leukemia (AML,CLL), non-Hodgkin lymphoma (NHL), esophageal cancer including cancer ofthe gastric-esophageal junction (OSCAR), gallbladder cancer andcholangiocarcinoma (GBC, CCC), urinary bladder cancer (UBC), uterinecancer (UEC), and preferably lung cancer cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”,that can be used in the diagnosis of cancer, preferably lung cancer(including NSCLC). The marker can be over-presentation of the peptide(s)themselves, or over-expression of the corresponding gene(s). The markersmay also be used to predict the probability of success of a treatment,preferably an immunotherapy, and most preferred an immunotherapytargeting the same target that is identified by the biomarker. Forexample, an antibody or soluble TCR can be used to stain sections of thetumor to detect the presence of a peptide of interest in complex withMHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Collagen alpha-3(VI) chain protein (COL6A3)—COL6A3 encodes the alpha-3chain, one of the three alpha chains of type VI collagen. The proteindomains have been shown to bind extracellular matrix proteins, aninteraction that explains the importance of this collagen in organizingmatrix components. Remodeling of the extracellular matrix throughover-expression of collagen VI contributes to cisplatin resistance inovarian cancer cells. The presence of collagen VI correlated with tumorgrade, an ovarian cancer prognostic factor (Sherman-Baust et al., 2003).COL6A3 is over-expressed in colorectal tumor (Smith et al., 2009a),salivary gland carcinoma (Leivo et al., 2005) and differentiallyexpressed in gastric cancer (Yang et al., 2007). COL6A3 was identifiedas one of seven genes with tumor-specific splice variants. The validatedtumor-specific splicing alterations were highly consistent, enablingclear separation of normal and cancer samples and in some cases even ofdifferent tumor stages (Thorsen et al., 2008).

Solute carrier family 6 (amino acid transporter), member 14(SLC6A14)—SLC6A14 encodes the solute carrier family 6, member 14(SLC6A14). SLC6A14 is an amino acid transporter and a member of thesolute carrier family 6. Members of this family are sodium and chloridedependent amino acid/neurotransmitter transporters. SLC6A14 transportsneutral and cationic amino acids. The transporter is expressed at lowlevels in normal tissues (Sloan and Mager, 1999). SLC6A14 was shown tobe up-regulated in cervical (Gupta et al., 2006), colorectal (Gupta etal., 2005) and estrogen receptor(ER)-positive breast cancer (Karunakaranet al., 2011) tissues and cell lines as well as hepatoma cells (Fuchs etal., 2004). While SLC6A14 is minimally expressed in the correspondingnormal tissues/cells, cancer cells up-regulate SLC6A14 to meet theirincreased demand for these amino acids. Alpha-methyl-DL-tryptophan(alpha-MT), a selective blocker of SLC6A14, induced amino aciddeprivation and caused apoptosis in ER-positive breast cancer cell lines(Karunakaran et al., 2011).

Dual specificity phosphatase 4 (DUSP4)—The protein encoded by the DUSP4gene is a member of the dual specificity protein phosphatase subfamily.DUSP4 inactivates ERK1, ERK2 and JNK, is expressed in a variety oftissues, and is localized in the nucleus. DUSP4 (alias MKP2) has beenreported to be over-expressed in malignant as compared to non-malignantbreast cancer samples (Wang et al., 2003). In colorectal cancer patientmicroarray datasets, DUSP4 expression was found to be differentiallyexpressed, with the highest expression in BRAF mutated tumors. Moreover,high DUSP4 was associated with a worse overall survival (De, V et al.,2013).

Glycoprotein (transmembrane) nmb (GPNMB)—The gene GPNMB encodes a type Itransmembrane glycoprotein. GPNMB has been shown to be expressed on alarge panel of different cancer types and to mainly increase tumoraggressiveness by promoting tumor cell migration, invasion andmetastasis formation. On the molecular level it was shown that GPNMBincreases the expression of MMP-2, 3 and 9 and is itself regulated byp53 (Metz et al., 2005; Metz et al., 2007; Rose et al., 2007; Fiorentiniet al., 2014). High levels of GPNMB further correlate with reducedoverall survival in SCLC, GBM and ccRCC (Qin et al., 2014; Li et al.,2014; Kuan et al., 2006).

Keratin, type II cytoskeletal 80 (KRT80)—KRT80 encodes the keratin 80(KRT80). KRT80 has been found in virtually all types of epithelia and isrelated to advanced tissue or cell differentiation. KRT80 containingintermediate filaments are located at the cell margins close to thedesmosomal plaques, and only in cells entering terminal differentiation,KRT80 adopts a cytoplasmic distribution (Langbein et al., 2010).

Structural maintenance of chromosomes 4 (SMC4)—The SMC4 protein is acore component of the condensin complex that plays a role in chromatincondensation and has also been associated with nucleolar segregation,DNA repair, and maintenance of the chromatin scaffold (Cervantes et al.,2006).

Solute carrier family 1 (glutamate/neutral amino acid transporter),member 4 (SLC1A4)—SLC1A4 is an amino acid transporter which mediatessodium-dependent exchange of small neutral amino acids (reviewed in(Kanai et al., 2013)). SLC1A4 was described to be expressed bysignificantly more esophageal adenocarcinomas as compared to squamouscell carcinomas (Younes et al., 2000). Expression of SLC1A4 in prostatecancer cells was shown to be increased in response to androgen treatment(Wang et al., 2013a).

Keratin 5 (KRT5), Keratin 6A (KRT6A), Keratin 6B (KRT6B), Keratin 6C(KRT6C)—KRT5, KRT6A, KRT6B and KRT6C are homologous keratin proteins,which are intermediate filament proteins. Keratins are extensively usedas marker proteins in tumor diagnostics, since their expression patternrelates to the tissue of origin of the malignancy (reviewed in(Karantza, 2011)). Under normal circumstances, KRT6A and KRT6B appear toinhibit cell migration by sequestering and thus inhibiting activity ofthe pro-migratory Src kinase. Whether this mechanism also works incancer cells has not been investigated (Rotty and Coulombe, 2012).KRT5/6 staining has been proposed as one of several markers todistinguish poorly differentiated adenocarcinoma from squamous cellcarcinoma in NSCLC (Zhao et al., 2014b; Xu et al., 2014). Pulmonaryneuroendocrine tumors are also negative for KRT5/6 (Zhang et al., 2014).

Chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated(CCL18)—This antimicrobial gene is one of several Cys-Cys (CC) cytokinegenes clustered on the q arm of chromosome 17. The cytokine encoded bythis gene displays chemotactic activity for naive T cells, CD4+ and CD8+T cells and nonactivated lymphocytes, but not for monocytes orgranulocytes. Up-regulation of CCL18 levels in both tumor tissue andblood has been described in cancer, and CCL18 serum levels have beenproposed as biomarker for several tumor types. In multiple cases, acorrelation with advanced tumor stages and poor prognosis has been shown(e.g. gastric cancer (Wu et al., 2013a), breast cancer (Chen et al.,2011; Narita et al., 2011), prostate cancer (Chen et al., 2014), bladdercancer (Urquidi et al., 2012)). Serum levels of CCL18 were increased inNSCLC patients as compared to healthy controls. In addition, increasedserum levels predicted a diminished survival time in patients withadenocarcinoma (Plones et al., 2012). CCL18 is part of a 12-proteinserum biomarker panel proposed for identification of NSCLC (Ostroff etal., 2010).

Matrix metallopeptidase 12 (macrophage elastase) (MMP12)—MMP12, alsoknown as human metalloelastase (HME) or macrophage metalloelastase (MME)is a zinc endopeptidase recognized for its ability to degrade elastin.Apart from that, it has a broad substrate range, extending to othermatrix proteins such as collagens, fibronectin, laminin, proteoglycans,and non-matrix proteins such as alpha-1-antitrypsin. In asthma,emphysema and chronic obstructive pulmonary disease (COPD), MMP12 maycontribute to alveolar destruction and airway remodeling (Cataldo etal., 2003; Wallace et al., 2008). MMP12 has been implicated inmacrophage migration, and as it can generate angiostatin fromplasminogen, it contributes to inhibition of angiogenesis (Chakrabortiet al., 2003; Chandler et al., 1996; Sang, 1998). Like othermetalloproteinases, MMP12 is involved in physiological processes likeembryogenesis, wound healing and the menstrual cycle (Chakraborti etal., 2003; Labied et al., 2009), but also in pathological processes oftissue destruction. Although data are based on low numbers of patientsin several cases, there is ample evidence in literature that MMP12 isfrequently over-expressed in cancer (Denys et al., 2004; Hagemann etal., 2001; Ma et al., 2009; Vazquez-Ortiz et al., 2005; Ye et al.,2008). However, data are controversial with respect to the impact ofMMP12 over-expression on clinical parameters and prognosis. While it maybe involved in matrix dissolution and, thus, metastasis, it can alsoinhibit tumor growth through production of angiostatin, which negativelyimpacts angiogenesis (Gorrin-Rivas et al., 2000; Gorrin Rivas et al.,1998; Kim et al., 2004). For lung cancer, consequences of MMP12expression are controversial. MMP12 over-expression in epithelial cellshas been reported in inflammation-triggered lung remodeling. MMP12up-regulation may play a role in emphysema-to-lung cancer transition (Quet al., 2009). Animal studies suggest that MMP12 expression by stroma ormacrophages suppresses growth of lung tumors (Acuff et al., 2006;Houghton et al., 2006). However, there are also reports that MMP12over-expression in lung tumors correlates with recurrence, metastaticdisease and shorter relapse-free survival after resection (Cho et al.,2004; Hofmann et al., 2005).

Lysosomal-associated membrane protein 3 (LAMP3)—LAMP3 is a type Itransmembrane protein found in the lysosomal compartment, with a smallcytoplasmic domain and a heavily glycosylated luminal domain (Wilke etal., 2012). Up-regulation of LAMP3 has been reported in several cancers,however, expression of LAMP3 by tumor cells themselves has not beendemonstrated. LAMP3(+) DCs have been detected specifically at theinvasive tumor margin forming clusters with proliferating T-lymphocytesand have thus been proposed to reflect a local anti-tumor immuneresponse, for example in renal cell carcinoma (Middel et al., 2010),esophageal squamous cell carcinoma (Liu et al., 2010), colorectalcarcinoma (Yuan et al., 2008; Sande) et al., 2005), as well as inmelanoma (Ladanyi et al., 2007). A meta-analysis of transcriptomics datasuggested that low levels of LAMP3 expression in lung cancer might beassociated with shorter overall survival (Lindskog et al., 2014).

Centromere protein N (CENPN)—The protein encoded by the CENPN gene formspart of the nucleosome-associated complex and is important forkinetochore assembly. CENPN recognizes a centromere-specific histonevariant (CENP-A), and is thus required to define the recruitment sitefor many other centromeric proteins (Carroll et al., 2009). Depletion ofCENPN and other proteins of the nucleosome-associated complex (NAC) doesnot impair bipolar spindle formation but leads to defects in chromosomecongression (McClelland et al., 2007). CENPN, together with other NACproteins, is recruited to DNA double-strand breaks, and thus the complexhas been proposed to play a role in DNA repair (Zeitlin et al., 2009).

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2)—The proteinencoded by this gene is a membrane-bound homodimeric enzyme that islocalized to the cisternae of the rough endoplasmic reticulum. Mutationsin the coding region of this gene are associated with Bruck syndrome.PLOD2 up-regulation has been described in colorectal cancer (Nicastri etal., 2014), multiple myeloma (Slany et al., 2014) and cervical cancer(Rajkumar et al., 2011), and has been associated with bone metastasisformation (Blanco et al., 2012). A correlation of elevated PLOD2expression with poor prognosis has been shown for glioblastoma (Dong etal., 2005) as well as for breast cancer (Gilkes et al., 2013) andhepatocellular carcinoma, where it was also associated with increasedtumor size and formation of intrahepatic metastasis (Noda et al., 2012).

Matrix metallopeptidase 1 (MMP1)—MMP1 is part of the matrixmetalloproteinase (MMP) family. In general, MMPs play an essential rolein regulation of vascular function, remodeling and angiogenesis. Throughdegradation of ECM and other extracellular molecules, they facilitatemigration and invasion of endothelial cells and vascular smooth musclecells, and influence vascular cell proliferation and apoptosis (Chen etal., 2013). MMP1 over-expression has been described for several cancertypes and has been associated with angiogenesis, invasion, and poorsurvival. For example, elevated MMP1 levels have been described as anindependent factor for survival in colon cancer (Langenskiold et al.,2013), and MMP1 expression in tumor and stroma is associated with tumorprogression and poor prognosis in breast cancer (Bostrom et al., 2011).MMP1 levels have been shown to be elevated in both plasma and tumortissue of lung cancer patients and associated with advanced stage anddecreased survival (Li et al., 2010b). A meta-analysis has confirmed anassociation of MMP1-1607 1G/2G polymorphism with increased risk ofdeveloping lung cancer (Xiao et al., 2012).

Keratin 10 (KRT10), Keratin 12 (KRT12), Keratin 13 (KRT13), Keratin 14(KRT14), Keratin 15 (KRT15), Keratin 16 (KRT16), Keratin 17 (KRT17),Keratin 19 (KRT19)—The homologous keratin proteins KRT10, KRT12, KRT13,KRT14, KRT15, KRT16, KRT17 and KRT19 are intermediate filament proteins.Some of the keratins have been associated with stem cell properties,such as KRT14 which is considered a cancer stem cell marker (Hatina andSchulz, 2012; Schalken and van, 2003). KRT15 is used as a marker foridentification and targeting of epidermal stem cells (Adhikary et al.,2013; Troy et al., 2011), and KRT17 is expressed in stem cells in thebasal layer of the hair bulge (Bragulla and Homberger, 2009). Expressionpatterns of the different keratins have been analyzed in various cancertypes, and both up- and down-regulation have been reported. For example,high levels of KRT17 have been associated with poor prognosis (Wang etal., 2013b; Escobar-Hoyos et al., 2014) and advanced stage (Kim et al.,2012). For KRT13, the majority of studies suggest down-regulation incancerous tissue (Hourihan et al., 2003; Ida-Yonemochi et al., 2012),and expression of KRT13 appears to be replaced with that of KRT17 duringsquamous cell transformation (Mikami et al., 2011). For KRT10 and KRT15,both up- and down-regulation in cancer have been demonstrated bydifferent studies. KRT19 is consistently reported to be over-expressedin many cancer types, and has been associated with metastasis and poorsurvival (Zong et al., 2012; Lee et al., 2012). KRT12 is expressed incorneal epithelia. The corneas display down-regulation of keratin 12which is considered a differentiation marker (Zhang et al., 2010b).

Mucin 16, cell surface associated (MUC16)—MUC16 is the largest ofseveral membrane-bound mucins. MUC16 is a single-pass transmembraneprotein with a heavily glycosylated extracellular domain. MUC16 is atumor-associated antigen that is cleaved from the surface of ovariancancer cells and shed into blood and used as a well-establishedbiomarker for monitoring the growth of ovarian cancer (Bafna et al.,2010). Increased MUC16 expression levels have been demonstrated in lungsquamous cell carcinoma (Wang et al., 2014). In addition, high MUC16serum levels have been correlated with shortened survival of NSCLCpatients (Yu et al., 2013; Cedres et al., 2011). Combined with otherbiomarkers, MUC16 may be part of a gene expression signature forsubtypes of lung cancer (Li et al., 2012).

Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)(ITGA2)—ITGA2 encodes the alpha subunit of a transmembrane receptor forcollagens and related proteins. A limited number of studies havereported dysregulation of ITGA2 in cancer, with evidence for bothelevated as well as decreased levels: In pancreatic ductaladenocarcinoma, ITGA2 was hypomethylated and over-expressed, andelevated expression was associated with poor survival (Nones et al.,2014). In contrast, down-regulation of ITGA2 has been shown for prostatecarcinoma (Shaikhibrahim et al., 2011). Decreased expression of ITGA2was associated with metastasis formation and poor survival in breast andprostate cancer (Ramirez et al., 2011).

Olfactomedin-like 2B (OLFML2B)—OLFML2B belongs to the family ofolfactomedin proteins, which are extracellular glycoproteins, mainlyinvolved in the differentiation of chemosensory cilia, earlyneurogenesis, dorsalization of neural tubes, neuromuscular signaling,exocytosis of synaptic vesicles ant the pathogenesis of glaucoma. OLFM2Btranscripts can be detected in a variety of different tissues in themouse, including lung, stomach and prostate, but are absent in liver(Furutani et al., 2005). The OLFML2B gene maps to chromosome 1q23.3,which was demonstrated to be a susceptibility locus for schizophrenia inlinkage studies (Puri et al., 2007).

Tetratricopeptide repeat domain 13 (TTC13)—TTC13 belongs to the familyof tetratricopeptide repeat (TPR) domain containing proteins. TPRdomains appear to be important for chaperone function, cell cycle,transcription an protein transport and TPR motif containing proteins areoften associated with multiprotein complexes (Blatch and Lassie, 1999).The TCC13 gene maps to chromosome 1q42.2. Chromosome 1q42.2-43 wasdescribed as locus of a putative predisposing gene for prostate cancerin one linkage analysis study (Berthon et al., 1998), but this could notbe confirmed for larger patient populations in further studies (Singh,2000; Gibbs et al., 1999).

Dedicator of cytokinesis 2 (DOCK2)—The protein encoded by the DOCK2 genebelongs to the CDM protein family. DOCK2 is known as important factorfor lymphocyte migration and chemotaxis. Exome and whole genomesequencing studies identified mutations within the DOCK2 gene incolorectal cancer, esophageal adenocarcinoma and intraductal papillarymucinous neoplasms of the pancreas (Yu et al., 2014; Dulak et al., 2013;Furukawa et al., 2011). Furthermore, DOCK2 was shown to bedifferentially expressed in pediatric astrocytoma samples and mighttherefore represent an interesting therapeutic target for this disease(Zhao et al., 2014a).

Poliovirus receptor-related 1 (herpesvirus entry mediator C)(PVRL1)—PVRL1 encodes an adhesion protein that plays a role in theorganization of adherent junctions and tight junctions in epithelial andendothelial cells. The PVRL1 gene maps to chromosome 11q23, a regionthat has been found to be amplified in adenoid cystic carcinoma (Zhanget al., 2013). With an important function in cell adhesion, PVRL1 hasbeen associated with regulation of cell invasive and migratoryproperties as well as with epithelial-mesenchymal transition, bothcrucial processes in tumor development. PVRL1 was identified as part ofa signature profile of the squamous cell carcinoma subtype of cervicalcancer (Imadome et al., 2010). PVRL1 expression was found to beincreased in thyroid tumors compared to normal thyroid tissue andfurther increased in papillary thyroid cancer (Jensen et al., 2010).PVRL1/2 expression is associated with a more favorable prognosis inacute myeloid leukemia (Graf et al., 2005).

FK506 binding protein 10, 65 kDa (FKBP10)—FK506-binding protein 10(FKBP10) belongs to the FKBP-type peptidyl-prolyl cis/trans isomerasefamily. It is located in endoplasmic reticulum and acts as molecularchaperones (Ishikawa et al., 2008; Patterson et al., 2000). It is highlyexpressed in lung development and can be reactivated in a coordinatedmanner with extracellular matrix proteins after lung injury (Pattersonet al., 2005).

ATP-binding cassette, sub-family C (CFTR/MRP), member 1 (ABCC1)—Theprotein encoded by the ABCC1 gene is a member of the superfamily ofATP-binding cassette (ABC) transporters. ABC proteins transport variousmolecules across extra- and intracellular membranes. ABCC1 plays animportant role as drug efflux pump, in both normal and tumor cells (Chenand Tiwari, 2011). Several studies have described over-expression ofABCC1 in different tumor types, and in many cases, an association ofABCC1 expression levels with tumor stage, metastasis, and poor prognosishas been found (e. g. in breast, prostate, and lung cancer) (Deeley etal., 2006). A study in Chinese patients identified a SNP in the ABCC1gene to increase the susceptibility for NSCLC (Yin et al., 2011).Another study has reported an association between ABCC1 SNPs andprogression-free survival of NSCLC patients (Lamba et al., 2014).

Arachidonate 15-lipoxygenase, type B (ALOX15B)—ALOX15B encodes a memberof the lipoxygenase family of structurally related nonheme irondioxygenases involved in the production of fatty acid hydroperoxides.The role that ALOX15B, more well-known as 15-LOX-2, and its enzymaticproduct, 15-S-hydroxyeicosatetraenoic acid (15S-HETE), play in tumordevelopment, has been most intensively studied in prostate cancer.Several studies have demonstrated that ALOX15B expression levels, aswell as levels of 15S-HETE production, are significantly decreased inprostate cancer as compared to normal tissue or cell lines (Hu et al.,2013; Shappell et al., 2001). In normal lung, ALOX15B expression isrestricted to type II pneumocytes. Expression is elevated in NSCLC, andan inverse correlation has been described between ALOX15B levels andtumor grade as well as tumor cell proliferation index (Gonzalez et al.,2004).

Sphingomyelin phosphodiesterase, acid-like 3B (SMPDL3B)—SMPDL3B is asphingomyelin phosphodiesterase expressed in podocytes, whose expressionhas been associated with diabetic kidney disease and focal segmentalglomerulosclerosis. Decreased expression of SMPDL3B in kidney diseasehas been associated with actin cytoskeleton remodeling and apoptosis(Merscher and Fornoni, 2014). The SMPDL3B gene maps to chromosome1p35.3.

Glutamine-fructose-6-phosphate transaminase 2 (GFPT2)—GFPT2 is involvedin neurite outgrowth, early neuronal cell development, neuropeptidesignaling/synthesis and neuronal receptor (Tondreau et al., 2008).Genetic variants in GFPT2 are associated with type 2 diabetes anddiabetic nephropathy (Zhang et al., 2004). Furthermore, association ofSNPs in GFPT2 suggests that the gene involved in modulation of oxidativepathway could be major contributor to diabetic chronic renalinsufficiency (Prasad et al., 2010). DNA methylation of the GFPT2 genewas validated in primary acute lymphoblastic leukemia (ALL) samples.Patients with methylation of multiple CpG islands had a worse overallsurvival (Kuang et al., 2008). GFPT2 plays a role in glutaminemetabolism and was observed to be more highly expressed in mesenchymalcell lines. Glutamine metabolism may play an important role in tumorprogression and inhibitors of cellular metabolic pathways may be a formof epigenetic therapy (Simpson et al., 2012).

DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5)—DDX5 (p68) is anATP-dependent RNA helicase which plays a role in splicing, rRNAprocessing and ribosome biogenesis, miRNA processing, as well as intranscriptional regulation. DDX5 is a transcriptional coactivator ofseveral factors which play a role in cancer development, such asandrogen receptor, p53, and Runx2. Over-expression of DDX5 has beendemonstrated for a number of different cancer types, as for examplecolorectal cancer, breast cancer, prostate cancer, glioma,hepatocellular carcinoma, and leukemia (Dai et al., 2014; Fuller-Pace,2013).

Enolase 1, (alpha) (ENO1)—The ENO1 gene encodes enolase-alpha (ENOA),one of three enolase proteins, the others being enolase-beta and -gamma,respectively. ENO1/ENOA over-expression has been demonstrated in manycancer types (Capello et al., 2011). ENOA is a metalloenzyme thatfunctions in glycolysis in the synthesis of phosphoenolpyruvate.Elevated ENOA levels have been correlated with poor survival in NSCLCpatients (Chang et al., 2006). Similarly, another study demonstratedup-regulation of ENO1 expression in a poor prognosis group of lungadenocarcinoma patients (Pernemalm et al., 2013). ENOA has beendemonstrated as a tumor-associated antigen, and anti-ENOA antibodies aswell as ENOA-specific T cells have been detected in pancreaticadenocarcinoma patients (Cappello et al., 2009). Autoantibodies to ENOAhave also been detected in NSCLC patients, and ENOA expression has beenshown to be increased in NSCLC tissue (He et al., 2007; Li et al.,2006).

Killer cell lectin-like receptor subfamily D, member 1 (KLRD1)—KLRD1,more well-known as CD94, associates with NKG2 molecules to form aheterodimer that is expressed on natural killer (NK) cells and cytotoxicT lymphocytes (CTLs). The inhibitory receptor KLRD1 (CD94):NKG2A wasshown to be over-expressed in tumor-infiltrating lymphocytes for examplein renal cell carcinoma and cervical cancer, which might contribute toan impaired anti-tumor immune response (Schleypen et al., 2003; Sheu etal., 2005). Similarly, over-expression of HLA-E, the KLRD1 (CD94):NKG2Aligand, on tumor cells might additionally contribute to tumor immuneescape (Bossard et al., 2012; Gooden et al., 2011).

Collagen, type XII, alpha 1 (COL12A1)—The COL12A1 gene encodes the alphachain of type XII collagen, a member of the FACIT (fibril-associatedcollagens with interrupted triple helices) collagen family. Type XIIcollagen is a homotrimer found in association with type I collagen, anassociation that is thought to modify the interactions between collagenI fibrils and the surrounding matrix (Oh et al., 1992). COL12A1 may beinvolved in basement membrane regulation providing specific molecularbridges between fibrils and other matrix components (Thierry et al.,2004). COL12A1 is expressed in heart, placenta, lung, skeletal muscleand pancreas (Dharmavaram et al., 1998), in a variety of connectivetissues including articular and epiphyseal cartilage (Gregory et al.,2001; Walchli et al., 1994; Watt et al., 1992). COL12A1 wasdown-regulated in tumors with high microsatellite instability whencompared to the stable group with low or null microsatellite instability(Ortega et al., 2010).

ATP-binding cassette, sub-family A (ABC1), member 13 (ABCA13)—In human,the ATP-binding cassette (ABC) family of transmembrane transporters hasat least 48 genes and 7 gene subfamilies. The predicted ABCA13 proteinconsists of 5,058 amino acid residues making it the largest ABC proteindescribed to date (Prades et al., 2002). Knight et al. determined thatABCA13 protein is expressed in mouse and human hippocampus and cortex,both regions relevant to schizophrenia and bipolar disorder (Knight etal., 2009). The ABCA13 gene maps to chromosome 7p12.3, a region thatcontains an inherited disorder affecting the pancreas (Shwachman-Diamondsyndrome) as well as a locus involved in T-cell tumor invasion andmetastasis (INM7), and therefore is a positional candidate for thesepathologies (Prades et al., 2002).

Cyclin B2 (CCNB2)—CCNB2 is one of several cyclins that associate withthe major cell cycle-regulatory kinase CDK1 (CDC2). Cyclin levels aretranscriptionally regulated over the cell cycle, providing differentlevels of activity and specificity to CDK1, thus controlling cell cycleprogression. Expression of cyclin B2 is regulated by the tumorsuppressor genes p53 and BRCA1 which act by repressing cyclin B2transcription (Quaas et al., 2012; De et al., 2011). CCNB2 up-regulationhas been described in several tumor types, such as cervical cancer(Espinosa et al., 2013; Rajkumar et al., 2011), bladder cancer (Lu etal., 2010), colorectal carcinoma (Park et al., 2007), astrocytoma (Liuet al., 2013), and glioblastoma (Hodgson et al., 2009). CCNB2 expressionlevels were associated with poor prognosis in breast cancer andidentified as an independent prognostic maker for survival (Shubbar etal., 2013). CCNB2 is over-expressed in NSCLC (Hofmann et al., 2004), andwas identified an independent predictor of poor prognosis in patientswith lung adenocarcinoma, but not squamous cell carcinoma (Takashima etal., 2014).

MutS homolog 6 (MSH6)—MSH6 encodes a member of the DNA mismatch repairMutS family. MSH proteins, including MSH6, recognize errors in thegenome sequence during replication, preventing the duplication of thedamaged strand and repairing single strand breaks (Conde-Perezprina etal., 2012). In several kinds of cancer, mutations in MSH6 and anerroneous DNA mismatch repair machinery (MMR) were described (e.g.colorectal cancers (Sameer et al., 2014; Vilar and Gruber, 2010; Silvaet al., 2009; Kastrinos and Syngal, 2007; Davidson, 2007), pancreaticcancer (Solomon et al., 2012), ovarian cancer (Xiao et al., 2014),breast cancer (Mandi et al., 2013)). PRP3 pre-mRNA processing factor 3homolog (S. cerevisiae) (PRPF3)—PRPF3 encodes the pre-mRNA processingfactor 3. PRPF3 mediates recruitment of the nuclear RNA decay machineryto the spliceosome (Nag and Steitz, 2012). PRPF3 is up-regulated inhepatocellular carcinoma through a fetal/cancer-specific splice variantof the transcription factor HNF4alpha (Niehof and Borlak, 2008).

Lysophosphatidylcholine acyltransferase 1 (LPCAT1)—LPCAT1 catalyzes theconversion of lysophosphatidyl-choline (LPC) to phosphatidylcholine. Inaddition, LPCAT1 is able to convert lyso-PAF (alkylated LPC) intoplatelet-activating factor (PAF). LPCAT1 over-expression has beendescribed in colorectal cancer (Mansilla et al., 2009), hepatocellularcarcinoma (Morita et al., 2013), breast cancer (Abdelzaher and Mostafa,2015), prostate cancer (Xu et al., 2013; Grupp et al., 2013; Zhou etal., 2012), and lung cancer (Wu et al., 2013b). LPCAT1 over-expressionpromoted cell proliferation, migration, and invasion in vitro (Morita etal., 2013).

Downstream neighbor of SON (DONSON)—DONSON encodes the downstreamneighbor of SON (DONSON). DONSON is a centrosomal protein whose levelsare regulated over the cell cycle, peaking during S-phase. DONSON isrequired for formation of a proper mitotic spindle, and appears to playa role in the DNA damage response (Fuchs et al., 2010). No cancerrelated literature is available.

Budding uninhibited by benzimidazoles 1 homolog beta (yeast)(BUB1B)—BUB1B encodes BUB1 mitotic checkpoint serine/threonine kinase B,a serine/threonine-protein kinase. It functions as a mitotic regulatorthat ensures accurate segregation of chromosomes through its role in themitotic checkpoint and the establishment of propermicrotubule-kinetochore attachments. Both up- and down-regulation ofBUB1B expression has been reported in various tumors. In general, moreliterature reports on over-expression of BUB1B in cancer, and anassociation with tumor progression and poor prognosis has also beendescribed, as for example in nasopharyngeal carcinoma (Huang et al.,2012a), tonsillar carcinoma (Hannisdal et al., 2010), breast cancer(Maciejczyk et al., 2013), epithelial ovarian cancer (Lee et al., 2009),and pancreatobiliary-type adenocarcinoma (Gladhaug et al., 2010).Similarly, reduced BUB1B protein was associated with longer survival inprostate cancer (Cirak et al., 2013).

Component of oligomeric Golgi complex 4 (COG4)—COG4 a component of anoligomeric protein complex involved in the structure and function of theGolgi apparatus. Interaction studies suggest that COG4 serves as a corecomponent of the complex and holds a crucial role in theassembly/function of the complex (Loh and Hong, 2004). The COG subunitsCOG4, 6, and 8, are capable of interacting with defined Golgi SNAREs andare involved in defining the specificity of vesicular sorting within theGolgi (Willett et al., 2013). Furthermore, the COG complex has beenshown to regulate the maintenance of Golgi glycosylation machinery(Pokrovskaya et al., 2011).

Proteasome (prosome, macropain) 26S subunit, non-ATPase, 14(PSMD14)—PSMD14 is a component of the 26S proteasome, a multiproteincomplex that degrades proteins targeted for destruction by the ubiquitinpathway. The PSMD14 protein within the 19S complex (19S cap; PA700) isresponsible for substrate deubiquitination during proteasomaldegradation (Spataro et al., 1997). Aberrant expression and dysfunctionof proteasome subunits have been involved in malignant transformationand in cell resistance to various cytotoxic drugs. Over-expression ofPSMD14 in mammalian cells affects cell proliferation and the response tocytotoxic drugs like vinblastine, cisplatin and doxorubicin (Spataro etal., 2002). Down-regulation of PSMD14 by siRNA transfection had aconsiderable impact on cell viability causing cell arrest in the G0-G1phase, ultimately leading to senescence (Byrne et al., 2010).

RAD54 homolog B (S. cerevisiae) (RAD54B)—DNA repair and recombinationprotein RAD54B is a protein that in humans is encoded by the RAD54Bgene. RAD54 binds to double-stranded DNA, and displays ATPase activityin the presence of DNA. The human RAD54B protein is a paralog of theRAD54 protein, which plays important roles in homologous recombination.Homologous recombination (HR) is essential for the accurate repair ofDNA double-strand breaks (DSBs) (Sarai et al., 2008). Knockdown ofRAD54B, a gene known to be somatically mutated in cancer, causeschromosome instability (CIN) in mammalian cells (McManus et al., 2009).RAD54B elevated gene expression is significantly associated with shortertime-to-progression and poor OS in GBM patients (Grunda et al., 2010).

Frizzled Family Receptor 1 (FZD1), Frizzled Family Receptor 2 (FZD2),Frizzled Family Receptor 7 (FZD7)—

The genes FZD2, FZD1 and FZD7 are all from the ‘frizzled’ gene family;members of this gene family encode 7-transmembrane domain proteins thatare receptors for Wnt signaling proteins. The expression of the FZD2gene appears to be developmentally regulated, with high levels ofexpression in fetal kidney and lung and in adult colon and ovary (Sagaraet al., 1998; Zhao et al., 1995). The FZD1 protein contains a signalpeptide, a cysteine-rich domain in the N-terminal extracellular region,7 transmembrane domains, and a C-terminal PDZ domain-binding motif. TheFZD1 transcript is expressed in various tissues, including lung as wellas heart, kidney, pancreas, prostate, and ovary (Sagara et al., 1998).The expression of frizzled 1 and 2 receptors was found to beup-regulated in breast cancer (Milovanovic et al., 2004). The FZD7protein contains an N-terminal signal sequence, 10 cysteine residuestypical of the cysteine-rich extracellular domain of Fz family members,7 putative transmembrane domains, and an intracellular C-terminal tailwith a PDZ domain-binding motif. FZD7 gene expressions may downregulateAPC function and enhance beta-catenin-mediated signals in poorlydifferentiated human esophageal carcinomas (Sagara et al., 1998; Tanakaet al., 1998).

Wingless-type MMTV integration site family, member 5A (WNTSA)—Ingeneral, Wnt5a regulates a variety of cellular functions, such asproliferation, differentiation, migration, adhesion and polarity(Kikuchi et al., 2012). It is expressed in undifferentiated humanembryonic stem cells (Katoh, 2008). WNTSA is classified as anon-transforming WNT family member whose role in carcinogenesis is stillambiguous. It exhibits tumor suppressor activities in some cancers(thyroid, brain, breast and colorectum), but is aberrantly up-regulatedin cancers of lung, stomach and prostate (Li et al., 2010a). OncogenicWNTSA activates canonical WNT signaling in cancer stem cells forself-renewal, and non-canonical WNT signaling at the tumor-stromalinterface for invasion and metastasis (Katoh and Katoh, 2007).Expression of WNTSA has been described for a variety of tumor entities.For example, abnormal protein expression of Wnt5a was observed in 28% ofprostate cancer where it promotes aggressiveness (Yamamoto et al.,2010). Furthermore, WNTSA over-expression is described to be associatedwith poor prognosis and/or increasing tumor grade in ovarian cancer(Badiglian et al., 2009), melanoma (Da Forno et al., 2008; Weeraratna etal., 2002), GBM (Yu et al., 2007), lung cancer (Huang et al., 2005) andpancreatic cancer (Ripka et al., 2007). In HCC, it seems that thecanonical Wnt signaling pathway contributes to tumor initiation and thenoncanonical signaling to tumor progression (Yuzugullu et al., 2009).

Fibroblast activation protein, alpha (FAP)—Fibroblast activation protein(FAP) is a type II integral membrane glycoprotein belonging to theserine protease family. The putative serine protease activity of FAPalpha and its in vivo induction pattern may indicate a role for thismolecule in the control of fibroblast growth or epithelial-mesenchymalinteractions during development, tissue repair, and epithelialcarcinogenesis (Scanlan et al., 1994). Most normal adult tissues andbenign epithelial tumors show little or no detectable FAP expression.However, FAP expression is detected in the stroma of over 90% ofmalignant breast, colorectal, lung, skin and pancreatic tumors,fibroblasts of healing wounds, soft tissue sarcomas, and some fetalmesenchymal cells. FAP has a potential role in cancer growth andmetastasis through cell adhesion and migration processes, as well asrapid degradation of ECM components. Thus, it is present on tumor cellsinvading the ECM and an endothelial cell involved in angiogenesis, butis not expressed in inactive cells of the same type (Dolznig et al.,2005; Kennedy et al., 2009; Rettig et al., 1993; Rettig et al., 1994;Scanlan et al., 1994; Zhang et al., 2010a).

Cyclin B1 (CCNB1)—CCNB1 encodes cyclin B1, one of several mitoticcyclins that associates with CDK1/CDC2 to promote mitotic progression.CNB1 over-expression has been described in numerous cancer types and wasassociated with tumor progression and poor prognosis, as for example incolorectal carcinoma (Li et al., 2003), breast cancer (Aaltonen et al.,2009; Agarwal et al., 2009), NSCLC (Cooper et al., 2009), and esophagealsquamous cell carcinoma (Huang et al., 2014). Also in gastric cancer,CCNB1 expression was associated with regional lymph node metastasis andpoor clinical prognosis (Begnami et al., 2010; Fujita et al., 2012).Antibodies directed against CCNB1 have been detected in patients withlung or prostate cancer and have been proposed as a biomarker for earlydetection of lung cancer (Egloff et al., 2005; Zhang et al., 2003).

ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 (ATP2A1),ATPase, Ca++ transporting, cardiac muscle, fast twitch 2 (ATP2A2)—Bothgenes (ATP2A1 and ATP2A2) encode SERCA Ca(2+)-ATPases. Sarcoplasmicreticulum (SR)1/ER calcium ATPases (SERCAs) are calcium pumps thatcouple ATP hydrolysis with calcium transport across the SR/ER membrane(MacLennan et al., 1997). SERCAs are encoded by three homologous genes:SERCA1 (ATP2A1), SERCA2 (ATP2A2), and SERCA3 (Wu et al., 1995). Someevidence has emerged to show that SERCA may also have a direct impact onthe processes of apoptosis, differentiation, and cell proliferation(Chami et al., 2000; Ma et al., 1999; Sakuntabhai et al., 1999).Mutations in ATP2A1, encoding SERCA1, cause some autosomal recessiveforms of Brody disease, characterized by increasing impairment ofmuscular relaxation during exercise (Odermatt et al., 1996). ATP2A2 isan ATPase associated with Darier's disease, a rare, autosomal dominanthereditary skin disorder characterized by abnormal keratinization andacantholysis (Huo et al., 2010). Germline alterations of ATP2A2 maypredispose to lung and colon cancer and an impaired ATP2A2 gene might beinvolved in carcinogenesis (Korosec et al., 2006). In a Small Cell LungCancer (H1339) and an Adeno Carcinoma Lung Cancer (HCC) cell line the ERCa2+-content was reduced compared to normal human bronchial epithelial.The reduced Ca2+-content correlated with a reduced expression of SERCA 2pumping calcium into the ER (Bergner et al., 2009). ATP2A2 could bepotential prognostic markers for colorectal cancer CRC patients. It wasdetected in circulating tumor cells (CTCs), and the postoperativerelapse was significantly correlated with gene over-expression (Huang etal., 2012b).

Fibronectin 1 (FN1)—FN1 encodes fibronectin, a glycoprotein present in asoluble dimeric form in plasma, and in a dimeric or multimeric form atthe cell surface and in extracellular matrix. It has been reported thatin most tumors, FN1 is predominantly expressed by cancer-associatedfibroblasts (CAFs) and endothelial cells, but not tumor cells (Berndt etal., 2010). Elevated levels of FN1 have been reported for some cancertypes and associated with poor prognosis or cancer progression, as forexample in gallbladder cancer (Cao et al., 2015), prostate cancer (vonet al., 2013), and renal cell carcinoma (Steffens et al., 2012; Waalkeset al., 2010). FN1 has also been implicated in the stimulation of lungcancer pathogenesis, including cell growth, chemoresistance andinhibition of apoptosis (reviewed in (Ritzenthaler et al., 2008)).

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3)—IGF2BP3 isa member of the insulin-like growth factor-II mRNA-binding proteinfamily, implicated in mRNA localization, turnover and translationalcontrol. The protein contains several KH (K-homologous) domains, whichare important in RNA binding and are known to be involved in RNAsynthesis and metabolism. Expression occurs mainly during embryonicdevelopment and has been described for some tumors. Thus, IGF2BP3 isconsidered to be an oncofetal protein (Liao et al., 2005). IGF2BP3 maypromote tumor cell proliferation by enhancing IGF-II protein synthesisand by inducing cell adhesion and invasion through stabilization of CD44mRNA (Findeis-Hosey and Xu, 2012). Moreover, IGF2BP3 expression has beenstudied in many human neoplasms with growing evidence that it mediatesmigration, invasion, cell survival and tumor metastasis (Jeng et al.,2009; Kabbarah et al., 2010; Li et al., 2011; Liao et al., 2011; Lu etal., 2011; Hwang et al., 2012; Samanta et al., 2012) and it might alsobe implicated in angiogenesis (Suvasini et al., 2011; Chen et al.,2012). In lung adenocarcinomas, a higher frequency of IGF2BP3 expressioncan be detected in moderately or poorly differentiated adenocarcinomas,which may be associated with an aggressive biological behavior(Findeis-Hosey et al., 2010; Beljan et al., 2012; Findeis-Hosey and Xu,2012).

Laminin, Gamma 2 (LAMC2)—

Laminins, a family of extracellular matrix glycoproteins, are the majornoncollagenous constituent of basement membranes. They have beenimplicated in a wide variety of biological processes including celladhesion, differentiation, migration, signaling, neurite outgrowth andmetastasis. The LAMC2 gene encodes the laminin-5 gamma2 chain, which ispart of laminin-5, one of the major components of the basement membranezone. LAMC2 was frequently up-regulation by promoter demethylation ingastric cancer (Kwon et al., 2011). LAMC2 was found to be over-expressedin angiotropic melanoma areas vs. avascular melanoma areas (Lugassy etal., 2009). LAMC2 is a biomarker of bladder cancer metastasis, and itsexpression level was associated with tumor grade (Smith et al., 2009b).LAMB3 and LAMC2 genes were co-expressed in 21 of 32 non-SCLC cell lines(66%) but only in one of 13 SCLC cell lines (8%). Co-expression of theLAMB3 and LAMC2 genes was also observed in all 4 cases of primarynon-SCLC cells examined but not in the corresponding non-cancerous lungcells (Manda et al., 2000).

Cerebral endothelial cell adhesion molecule (CERCAM)—CERCAM is localizedat the surface of endothelial cells (Starzyk et al., 2000) and mapped onchromosome 9q34.11, a candidate region on 9q, identified as linked tofamilial idiopathic scoliosis (Miller et al., 2012). The CEECAM1 gene iswidely transcribed in the nervous system and in several secretorytissues such as salivary glands, pancreas, liver and placenta (Schegg etal., 2009). The CERCAM protein is structurally similar to the ColGaITenzymes GLT25D1 and GLT25D2. But although its function is still notknown, it seems to be is functionally different from the related GLT25D1protein, and the protein does not function as a glycosyltransferase likeGLT25D1 and GLT25D2 proteins (Perrin-Tricaud et al., 2011).

Matrix-remodeling associated 5 (MXRA5)—MXRA5, also known as adlican,encodes an adhesion proteoglycan and belongs to a group of genesinvolved in ECM remodeling and cell-cell adhesion (Rodningen et al.,2008). Although the function of MXRA5 in cancer is unknown, somaticmutations in MXRA5 have been identified in tumors obtained from avariety of tissues such as skin, brain, lung, and ovary. RT-PCR wasperformed on adlican (MXRA5) confirmed microarray findings ofover-expression in colon cancers compared to normal colon tissue (13colorectal tumors and 13 normal tissues) (Zou et al., 2002). In a recentstudy, matrix-remodeling associated 5 was the second most frequentlymutated gene in NSCLC (first is TP53) (Xiong et al., 2012).

ADAM metallopeptidase domain 8 (ADAM8)—ADAM8 is a member of the ADAM (adisintegrin and metalloprotease domain) family. Many ADAM species,including ADAM8, are expressed in human malignant tumors, where they areinvolved in the regulation of growth factor activities and integrinfunctions, leading to promotion of cell growth and invasion (Mochizukiand Okada, 2007). The expression of ADAM8 was positively correlated toEGFR. Both were mainly expressed in the cytoplasm and on the cellmembrane (Wu et al., 2008). ADAM8 was abundantly expressed in the greatmajority of lung cancers examined. Exogenous expression of ADAM8increased the migratory activity of mammalian cells, an indication thatADAM8 may play a significant role in progression of lung cancer(Ishikawa et al., 2004). ADAM8 has been associated with poor prognosisof lung cancer (Hernandez et al., 2010). ADAM8 over-expression wasassociated with shorter patient survival and it was a good predictor ofdistant metastases in RCC (Roemer et al., 2004b; Roemer et al., 2004a).In addition, expression levels and the protease activities of ADAM8correlated with invasive activity of glioma cells, indicating that ADAM8may play a significant role in tumor invasion in brain cancer (Wildeboeret al., 2006).

Melanoma antigen family F, 1 (MAGEF1)—Most known members of the MAGE(melanoma-associated antigen) superfamily are expressed in tumors,testis and fetal tissues, which has been described as a cancer/testisexpression, pattern (MAGE subgroup I). Peptides of MAGE subgroup I havebeen successfully used in peptide and DC vaccination (Nestle et al.,1998; Marchand et al., 1999; Marchand et al., 1999; Marchand et al.,1995; Thurner et al., 1999). In contrast, some MAGE genes (MAGE subgroupII), such as MAGEF1, are expressed ubiquitously in all adult and fetaltissues tested and also in many tumor types including ovarian, breast,cervical, melanoma and leukemia (Nestle et al., 1998; Marchand et al.,1999; Marchand et al., 1999; Marchand et al., 1995; Thurner et al.,1999). Nevertheless, over-expression of MAGEF1 could be detected inNSCLC (Tsai et al., 2007) and in 79% of a cohort of Taiwanese colorectalcancer patients (Chung et al., 2010).

Small nuclear ribonucleoprotein 200 kDa (U5) (SNRNP200)—Pre-mRNAsplicing is catalyzed by the spliceosome, a complex of specialized RNAand protein subunits that removes introns from a transcribed pre-mRNAsegment. The spliceosome consists of small nuclear RNA proteins (snRNPs)U1, U2, U4, U5 and U6, together with approximately 80 conservedproteins. SNRNP200 is a gene required for unwinding of the U4/U6 duplex,a step essential for catalytic activation of the spliceosome (Maeder etal., 2009). SNRNP200 expression was detected in heart, brain, placenta,lung, liver, skeletal muscle, kidney, and pancreas (Zhao et al., 2009a).Mutations in SNRNP200 have recently been discovered to be associatedwith autosomal dominant retinitis pigmentosa (adRP) (Benaglio et al.,2011; Liu et al., 2012).

TPX2, microtubule-associated, homolog (Xenopus laevis) (TPX2)—TPX2 is aspindle assembly factor. It is required for normal assembly of mitoticspindles and of microtubules during apoptosis. TPX2 is required forchromatin and/or kinetochore dependent microtubule nucleation (Bird andHyman, 2008; Moss et al., 2009). Newly synthesized TPX2 is required fornearly all Aurora A activation and for full p53 synthesis andphosphorylation in vivo during oocyte maturation (Pascreau et al.,2009). TPX2 is a cell cycle-associated protein which is over-expressedin many tumor types, such as meningiomas (Stuart et al., 2010), squamouscell carcinoma of the larynx (SCCL) (Cordes et al., 2010), oral squamouscell carcinomas (SCC) (Shigeishi et al., 2009), hepatocellularcarcinomas (HCC) (Satow et al., 2010), pancreatic tumor (Warner et al.,2009), ovarian cancer (Ramakrishna et al., 2010), squamous cellcarcinoma of the lung (Lin et al., 2006; Ma et al., 2006). It isfrequently co-over-expressed with Aurora-A giving rise to a novelfunctional unit with oncogenic properties (Asteriti et al., 2010). TPX2expression is a prognostic indicator in lung cancer (Kadara et al.,2009).

Transforming growth factor, beta-induced, 68 kDa (TGFBI)—TGFBI was firstidentified as a TGF-beta-inducible gene in a human lung adenocarcinomacell line. It encodes for a secreted extracellular matrix protein, whichis thought to act on cell attachment and extracellular matrixcomposition. Normally, the expression of TGFBI is mainly found infibroblasts, keratinocytes, and muscle cells (Bae et al., 2002). TGFBIis over-expressed in several solid tumors such as colon (Kitahara etal., 2001), pancreas (Schneider et al., 2002) and kidney (Ivanov et al.,2008). TGFBI is down-regulated in lung cancer (Zhao et al., 2004; Shaoet al., 2006), reduces the metastatic potential of lung tumor cells (Wenet al., 2011) and when over-expressed, contributes to apoptotic celldeath (Zhao et al., 2006). In NSCLC samples a strong association betweenelevated TGFBI expression and the response to chemotherapy was observed(Irigoyen et al., 2010).

Cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6)—CDK4is a member of the Ser/Thr protein kinase family. It is a catalyticsubunit of the protein kinase complex that is important for cell cycleG1 phase progression. The activity of this kinase is restricted to theG1- to S phase transition during the cell cycle and its expression isprimarily controlled at the transcriptional level (Xiao et al., 2007).CDK4 and CDK6 enzymes and their regulators, e.g., cyclins, play criticalroles in embryogenesis, homeostasis, and cancerogenesis (Graf et al.,2010). In lung cancer tissues the expression level of CDK4 protein wassignificantly increased compared to normal tissues (P<0.001). Patientswith higher CDK4 expression had a markedly shorter overall survival timethan patients with low CDK4 expression. Multivariate analysis suggestedthe level of CDK4 expression was an independent prognostic indicator(P<0.001) for the survival of patients with lung cancer. Furthermore,suppressing CDK4 expression also significantly elevated the expressionof cell cycle regulator p21 (Wu et al., 2011). In lung cells thatexpress an endogenous K-Ras oncogene, ablation of Cdk4, but not Cdk2 orCdk6, induces an immediate senescence response. No such response occursin lungs expressing a single Cdk4 allele or in other K-Ras-expressingtissues. Targeting Cdk4 alleles in advanced tumors detectable bycomputed tomography scanning also induces senescence and prevents tumorprogression (Puyol et al., 2010).

Versican (VCAN)—VCAN is a member of the aggrecan/versican proteoglycanfamily. VCAN is known to associate with a number of molecules in theextracellular matrix including hyaluronan, tenascin, fibulin-1,fibronectin, CD44 and L-selectin, fibrillin, integrin, and link protein(Zheng et al., 2004). VCAN is expressed in a variety of tissues. It ishighly expressed in the early stages of tissue development, and itsexpression decreases after tissue maturation. Its expression is alsoelevated during wound repair and tumor growth (Ghosh et al., 2010).Knockdown in human lung adenocarcinoma (A549) cells of VCAN by RNAinterference significantly inhibited tumor growth in vivo but not invitro (Creighton et al., 2005). VCAN is a direct target of p53. Highexpression of VCAN has also been found in the peritumoral stromal tissueof early stage prostate cancers, and of breast cancers, and it isassociated with an aggressive tumor behavior (Yoon et al., 2002).

Ubiquitin-conjugating enzyme E2S (UBE2S)—UBE2S is an APC auxiliaryfactor that promotes mitotic exit. Its depletion prolongs drug-inducedmitotic arrest and suppresses mitotic slippage (Garnett et al., 2009).UBE2S is over-expressed in common human cancers. In esophageal cancer,UBE2S is significantly associated with the extent of tumor burden. Itspositivity was linked to poor response to neoadjuvant therapy and worsesurvival (Chen et al., 2009). In the UBE2S promoter, binding sites forearly growth response-1 (Egr-1) and serum response factor (SRF) wereidentified. Over-expression of these factors increased UBE2S expressionwhich was required for cancer cell proliferation (Lim et al., 2008).

SET and MYND domain containing 3 (SMYD3)—It was previously reported thatup-regulation of SMYD3, a histone H3 lysine-4-specificmethyltransferase, plays a key role in the proliferation of colorectalcarcinoma (CRC) and hepatocellular carcinoma (HCC). In another study,they reveal that SMYD3 expression is also elevated in the great majorityof breast cancer tissues. Similarly to CRC and HCC, silencing of SMYD3by small interfering RNA to this gene resulted in the inhibited growthof breast cancer cells, suggesting that increased SMYD3 expression isalso essential for the proliferation of breast cancer cells (Hamamoto etal., 2006). Knockdown of SMYD3 by RNA interference down-regulates c-Metexpression and inhibits cells migration and invasion induced by HGF (Zouet al., 2009). SMYD3 plays crucial roles in HeLa cell proliferation andmigration/invasion, and it may be a useful therapeutic target in humancervical carcinomas (Wang et al., 2008).

Dystonin (DST)—

DST (BPAG1-e) encodes a member of the plakin protein family of adhesionjunction plaque proteins. BPAG1-e is expressed in epithelial tissue,anchoring keratin-containing intermediate filaments to hemidesmosomes(HDs). HDs are multiprotein adhesion complexes that promote epithelialstromal attachment in stratified and complex epithelia. Modulation oftheir function is of crucial importance in a variety of biologicalprocesses, such as differentiation and migration of keratinocytes duringwound healing and carcinoma invasion, in which cells become detachedfrom the substrate and acquire a motile phenotype (Litjens et al.,2006). Malignant melanoma is one of the most aggressive types of tumor.BPAG1 is expressed in human melanoma cell lines (A375 and G361) andnormal human melanocytes. The levels of anti-BPAG1 auto-antibodies inthe sera of melanoma patients were significantly higher than in the seraof healthy volunteers (p<0.01). Anti-BPAG1 auto-antibodies may be apromising marker for the diagnosis of melanoma (Shimbo et al., 2010).DST was associated with breast cancer invasion (Schuetz et al., 2006).The BPAG1 gene is likely to be involved in the proliferation, apoptosis,invasion and metastasis of nasopharyngeal carcinoma NPC (Fang et al.,2005).

Solute carrier family 34 (sodium phosphate), member 2 (SLC34A2)—SLC34A2is a pH-sensitive sodium-dependent phosphate transporter. Up-regulationof SLC34A2 gene expression in well-differentiated tumors may reflectcell differentiation processes during ovarian cancerogenesis and couldserve as potential marker for ovarian cancer diagnosis and prognosis(Shyian et al., 2011). RT-PCR confirmed increased expression of SLC34A2in papillary thyroid cancer (Kim et al., 2010b). There was also asignificantly increased gene expression of SLC34A2 among breast cancertissues compared with normal tissues (Chen et al., 2010).

Tenascin C (hexabrachion) (TNC)—Tenascin-C (TNC) is an extracellularmatrix protein that is highly up-regulated in processes that are closelyassociated with elevated migratory activity such as embryonicdevelopment (Bartsch et al., 1992), wound healing (Mackie et al., 1988)and neoplastic processes (Chiquet-Ehrismann, 1993; Chiquet-Ehrismann andChiquet, 2003). Furthermore, TNC is over-expressed in tumor vessels thathave a high proliferative index, which indicates that TNC is involved inneoplastic angiogenesis (Kim et al., 2000). Over-expression of TNC hasfurther been reported from colon cancer (De et al., 2013), adenoidcystic carcinoma, where it has been associated with worst prognosis (Siuet al., 2012), juvenile nasopharyngeal angiofibroma, where it possiblypromotes angiogenesis (Renkonen et al., 2012), advanced melanoma(Fukunaga-Kalabis et al., 2010), pancreatic cancer, where it plays arole in proliferation, migration and metastasis (Paron et al., 2011).

Reticulocalbin 1, EF-hand calcium binding domain (RCN1), reticulocalbin3, EF-hand calcium binding domain (RCN3)—Reticulocalbin 1 is acalcium-binding protein located in the lumen of the ER.Immunohistochemical examination demonstrated a broad distribution of RCNin various organs of fetuses and adults, predominantly in the endocrineand exocrine organs. Over-expression of RCN may play a role intumorigenesis, tumor invasion, and drug resistance (Fukuda et al.,2007). Reticulocalbin 1 (RCN1) is a cell surface-associated protein onboth endothelial (EC) and prostate cancer (PCa) cell lines. RCN1expression on the cell surface was up-regulated by tumor necrosis factoralpha treatment of bone-marrow endothelial cells (Cooper et al., 2008).RCN1 is up-regulated in colorectal carcinoma (CRC) and was localized incancer cells or in stromal cells near the cancer cells. It could be anovel candidate for CRC marker (Watanabe et al., 2008). RCN3 is a memberof the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multipleEF-hand Ca2+-binding proteins localized to the secretory pathway (Tsujiet al., 2006). In oligodendrogliomas RCN3 is suggested as a potentiallyimportant candidate gene. Though little is known about the function ofRCN3 (Drucker et al., 2009).

Basonuclin 1 (BNC1)—Basonuclin is a zinc-finger protein with a highlyrestricted tissue distribution (Tseng, 1998). Thus far, basonuclin hasbeen detected mainly in the basal keratinocytes of stratified squamousepithelia (skin, oral epithelium, esophagus, vagina, and cornea) and inthe gametogenic cells of the testis and ovary (Tseng and Green, 1994;Weiner and Green, 1998). There is now considerable evidence thatbasonuclin is a cell-type-specific transcription factor for rRNA genes(rDNA). The zinc fingers of basonuclin interact with threeevolutionarily conserved sites within the rDNA promoter (luchi andGreen, 1999; Tseng et al., 1999). Epigenetic regulation by CpGmethylation has an important role in tumorigenesis as well as in theresponse to cancer therapy. BNC1 was hypomethylated in radioresistantH1299 human non-small cell lung cancer (NSCLC) cell lines. Suppressionof BNC1 mRNA expression in H1299 cells also reduced the resistance ofthese cells to ionizing radiation (Kim et al., 2010a). Aberrant DNAmethylation of BNC1 was also detected in chronic lymphocytic leukemia(CLL) samples (Tong et al., 2010). In Renal Cell Carcinoma (RCC),methylation of BNC1 was associated with a poorer prognosis independentof tumor size, stage or grade (Morris et al., 2010).

Transforming, acidic coiled-coil containing protein 3 (TACC3)—TACC3exists in a complex with ch-TOG (colonic and hepatic tumorover-expressed gene) and clathrin that cross-links microtubules inkinetochore fibers. TACC3 is expressed in certain proliferative tissuesincluding testis, lung, spleen, bone marrow, thymus and peripheral bloodleukocytes. TACC3 expression is altered in some human tumor types. Incells, TACC3 is localized to both centrosomes and spindle microtubulesbut not at astral microtubules (Hood and Royle, 2011). TACC3 expressionwas correlated with p53 expression, and patient whose tumors highlyexpressed TACC3 and p53 had a significantly poorer prognosis thanpatients whose tumors had low-level expression for both immunostainings(P=0.006). It is suggested that increase in TACC3 may impart aproliferative advantage to NSCLC and contribute to tumor progression,and that TACC3 expression is a strong prognostic indicator of clinicaloutcome in NSCLC (Jung et al., 2006). Tacc3 may be a negative regulatorof the Notch signaling pathway (Bargo et al., 2010).

Pecanex-like 3 (Drosophila) (PCNXL3)—Pecanex-like protein 3 (PCNXL3) isa multi-pass membrane protein; it belongs to the pecanex family. ThePCNXL3 gene was mapped to the chromosomal region 11q12.1-q13. Threenovel human tumor-associated translocation breakpoints were located inthe chromosome 11q13 region between the markers D1154933 and D115546.Thus PCNXL3 might be an 11q13-associated disease gene (van et al.,2000).

Drosha, ribonuclease type III (DROSHA)—Drosha is a Class 2 RNase IIIenzyme responsible for initiating the processing of microRNA (miRNA), orshort RNA molecules naturally expressed by the cell that regulate a widevariety of other genes by interacting with the RNA-induced silencingcomplex (RISC) to induce cleavage of complementary messenger RNA (mRNA)as part of the RNAi pathway. A microRNA molecule is synthesized as along RNA primary transcript known as a pri-miRNA, which is cleaved byDrosha to produce a characteristic stem-loop structure of about 70 basepairs long, known as a pre-miRNA (Lee et al., 2003). Drosha exists aspart of a protein complex called the Microprocessor complex, which alsocontains the double-stranded RNA binding protein Pasha (also calledDGCR8) (Denli et al., 2004), which is essential for Drosha activity andis capable of binding single-stranded fragments of the pri-miRNA thatare required for proper processing (Han et al., 2006). Human Drosha wascloned in 2000, when it was identified as a nuclear dsRNA ribonucleaseinvolved in the processing of ribosomal RNA precursors (Wu et al.,2000). Drosha was the first human RNase III enzyme identified andcloned. The other two human enzymes that participate in the processingand activity of miRNA are the Dicer and Argonaute proteins. Both Droshaand Pasha are localized to the cell nucleus, where processing ofpri-miRNA to pre-miRNA occurs. This latter molecule is then furtherprocessed by the RNase Dicer into mature miRNAs in the cell cytoplasm(Lee et al., 2003). Drosha and other miRNA processing enzymes may beimportant in cancer prognosis (Slack and Weidhaas, 2008)

Cell division cycle 6 homolog (S. cerevisiae) (CDC6)—CDC6 proteinfunctions as a regulator at the early steps of DNA replication. Itlocalizes in cell nucleus during cell cycle G1, but translocates to thecytoplasm at the start of S phase. Further, CDC6 is supposed to regulatereplication-checkpoint activation through the interaction with ATR inhigher eukaryotic cells (Yoshida et al., 2010). CDC6 is essential forDNA replication and its de-regulation is involved in carcinogenesis. Itwas found that CDC6 down-regulation by RNA interference (RNAi) preventedcell proliferation and promoted apoptosis (Lau et al., 2006).Over-expression of CDC6 was found in several cancers. Among the cancertypes over-expressing CDC6 are gastric cancer (Tsukamoto et al., 2008),brain tumors (Ohta et al., 2001), oral squamous cell carcinoma (Feng etal., 2008), cervical carcinoma (Wang et al., 2009) and malignantmesothelioma (Romagnoli et al., 2009).

Deiodinase, iodothyronine, type II (DI02)—The protein encoded by theD102 gene belongs to the iodothyronine deiodinase family. It is highlyexpressed in the thyroid, and may contribute significantly to therelative increase in thyroidal T3 production in patients with Gravesdisease and thyroid adenomas (Meyer et al., 2008); (de Souza Meyer etal., 2005)). The gene expression patterns are significantly differentbetween upward, and downward progressing types of nasopharygealcarcinoma (NPC). The expression of D102 gene is higher in the downwardprogressing type (downward=distant metastasis) than in upwardprogressing type (local growth and invasion of the base of skull), whichmay be closely related to the metastasis potential of NPC (Liang et al.,2008a). D102 mRNA as well as D102 activity are expressed in brain tumors(Murakami et al., 2000). D2 activity in lung is present and similar inperipheral lung and lung cancer tissue (Wawrzynska et al., 2003).

Kinesin family member 26B (KIF26B)—A kinesin is a protein belonging to aclass of motor proteins found in eukaryotic cells. Kinesins move alongmicrotubule filaments, and are powered by the hydrolysis of ATP (thuskinesins are ATPases). Kif26b, a kinesin family gene, is a downstreamtarget of Sall1 (Nishinakamura et al., 2011). Kif26b is essential forkidney development because it regulates the adhesion of mesenchymalcells in contact with ureteric buds. Over-expression of Kif26b in vitrocaused increased cell adhesion through interactions with non-musclemyosin (Terabayashi et al., 2012; Uchiyama et al., 2010).

Serpin peptidase inhibitor, clade B (ovalbumin), member 3(SERPINB3)—Squamous cellular carcinoma antigen (SCCA), also calledSERPINB3, is a member of the high molecular weight family of serineprotease inhibitors (serpins) (Suminami et al., 1991). High levels havebeen reported in cancer of the head and neck tissue and other epithelialcancers (Torre, 1998). SCCA has been reported to be over-expressed intumoral compared to peritumoral tissue, suggesting a role as a potentialmarker for histological detection of HCC (Pontisso et al., 2004). SerpinB3/B4, particularly Serpin B4, appears to play an important role inaberrant epithelial proliferation. Evaluation of Serpin B3/B4 could haveprognostic value in predicting disease progression, especially inpatients with increased susceptibility to lung cancer (Calabrese et al.,2012). On one hand, SCCA1 (SERPINB3) inhibits cell death induced bylysosomal injury while, on the other hand, it sensitizes cells to ERstress by activating caspase-8 independently of the death receptorapoptotic pathway (Ullman et al., 2011). Some findings indicate thatSERPINB3 plays an important role in the induction of epidermal barrierdisruption. SERPINB3 may be a critical determinant of barrier functionin the epidermis (Katagiri et al., 2010).

Cyclin-dependent kinase 1 (CDK1)—CDC2 (cell division cycle 2), alsoknown as p34cdc² or CDK1 (Cyclin-dependent kinase 1), belongs to theCDKs, a family of serine/threonine protein kinases, and plays a key rolein cell cycle control (Vermeulen et al., 2003). Over-expression of CDC2was found in several cancers, although according to (Vermeulen et al.,2003) the expression of other cell cycle proteins such as cyclins isdysregulated even more frequently. Over-expression of CDC2 has beendescribed for NSCLC (Xu et al., 2011; Zhang et al., 2011). Perumal etal. (2012) reported that over-expression of CDC2 correlated with poorprognosis (Perumal et al., 2012). Furthermore, a study suggested thepossible clinical use of CDC2 as a predictor of recurrence inearly-stage non-small cell lung cancer (Kubo et al., 2014).

Collagen, type XI, alpha 1 (COL11A1)—COL11A1 encodes one of the twoalpha chains of type XI collagen, a minor fibrillar collagen. COL11A1was reported to be up-regulated several cancers, e.g. in colorectalcancer (Freire et al., 2014), in breast cancer (Ellsworth et al., 2009),in gastric cancer (Zhao et al., 2009b), bladder tumors (Ewald et al.,2013). COL11A1 expression in ovarian cancer was linked to cancerrecurrence and poor survival. Knockdown of COL11A1 decreased in vitrocell migration, invasion, and tumor progression in mice (Cheon et al.,2014; Wu et al., 2014b). COL11A1 was found to be differentiallyexpressed lung tissue of nonsmoking female lung cancer patients ascompared to healthy controls, based on microarray analysis (Lv and Wang,2015).

Collagen, type I, alpha 2 (COL1A2)—COL1A2 encodes the pro-alpha2 chainof type I collagen whose triple helix comprises two alpha1 chains andone alpha2 chain. In gastric cancer samples COL1A2 was found to beup-regulated as compared to normal tissue (Yan et al., 2014; Yang etal., 2007) and associated with advanced stage (Yasui et al., 2004).COL1A2 was reported to be up-regulated in osteosarcoma (Wu et al.,2014a), in advanced stage bladder cancer (Fang et al., 2013), In headand neck/oral squamous cell carcinoma (HNOSCC) (Ye et al., 2008), and inmedulloblastoma, the most common malignant brain tumor of children(Liang et al., 2008b).

Periostin, osteoblast specific factor (POSTN)—POSTN, a gene encoding aprotein with similarity to the fasciclin family and involved in cellsurvival and angiogenesis, has emerged as a promising marker for tumorprogression in various types of human cancers (Ruan et al., 2009). Highexpression of periostin protein or mRNA was detected in most solidtumors including breast (Zhang et al., 2010c), colon (Kikuchi et al.,2008), head and neck (Kudo et al., 2006), pancreatic (Kanno et al.,2008), papillary thyroid (Puppin et al., 2008), prostate (Tischler etal., 2010), ovarian (Choi et al., 2010), lung (Takanami et al., 2008)and liver (Utispan et al., 2010) carcinoma, as well as esophagealsquamous cell carcinoma (Kwon et al., 2009). Periostin is abnormallyhighly expressed in lung cancer and is correlated with angiogenesis,invasion and metastasis (Takanami et al., 2008). Silencing of periostinin A549 non-small cell lung cancer (NSCLC) cells inhibits tumor cellgrowth and decrease cell invasion (Wu et al., 2013c).

AT hook, DNA binding motif, containing 1 (AHDC1)—This gene encodes aprotein containing two AT-hooks, which likely function in DNA binding.Mutations in this gene were associated with cerebral visual impairment(Bosch et al., 2015). Using whole-exome sequencing, AHDC1 de novotruncating mutations were identified in individuals with syndromicexpressive language delay, hypotonia, and sleep apnea. The mutationsmost likely cause this genetic syndrome (Xia et al., 2014).

Apoptosis-inducing factor, mitochondrion-associated, 2 (AIFM2)—This geneencodes a flavoprotein oxidoreductase that binds single stranded DNA andis thought to contribute to apoptosis in the presence of bacterial andviral DNA. AIFM2 is not well characterized, but limited evidencesuggests that it may function as a tumor suppressor. AIFM2 expression isactivated by the tumor suppressor p53, and ectopic expression of p53 hasbeen demonstrated to induce apoptosis. Morever, AIFM2 expression wasshown to be downregulated in a panel of human tumors including kidney,stomach, colorectal, and other cancer samples (Ohiro et al., 2002; Wu etal., 2004). In a knockout mouse model, however, AIFM2 was not requiredfor p53-dependent tumor suppression (Mei et al., 2006). In cell culture,AIFM2 is involved in mediating adenosine-induced apoptosis (Yang et al.,2011).

Chromosome 6 open reading frame 132 (C6orf132)—C6orf132 encodes thechromosome 6 open reading frame 132. The gene C6orf132 is located onchromosome 6p21.1 (Mungall et al., 2003). The function of this gene isstill unknown. CCZ1 vacuolar protein trafficking and biogenesisassociated homolog (S. cerevisiae) (CCZ1), CCZ1 vacuolar proteintrafficking and biogenesis associated homolog B (S. cerevisiae)(CCZ1B)—CCZ1 encode CCZ1 vacuolar protein trafficking and biogenesisassociated homolog (S. cerevisiae). CCZ1B encode CCZ1 vacuolar proteintrafficking and biogenesis associated homolog B (S. cerevisiae). CCZ1and CCZ1B were identified as human genes evolutionarily conserved inCaenorhabditis elegans by comparative proteomics (Lai et al., 2000). Thegenes CCZ1 and CCZ1 B are located on chromosome 7p22.1 (Hillier et al.,2003). CCZ1 seems to act in lysosome biogenesis and phagosome maturationby recruiting the GTPase RAB7A7 over the phagosome (Nieto et al., 2010).The CCZ1 B gene is an uncharacterized gene.

Collagen, type V, alpha 2 (COL5A2)—This gene encodes an alpha chain forone of the low abundance fibrillar collagens. COL5A2 was reported to beup-regulated in colorectal cancer tissue samples as compared to adjacentnoncancerous tissues (Fischer et al., 2001). Matched samples of ductalcarcinoma in situ (DCIS), invasive ductal carcinoma (IDC) and stroma ofbreast cancer patients revealed elevated expression of COL5A2 in IDC(Vargas et al., 2012). In osteosarcoma, COL5A2 was reported to beup-regulated and to be important in tumorigenesis (Wu et al., 2014).

Collectin sub-family member 12 (COLEC12)—This gene encodes a member ofthe C-lectin family, proteins that possess collagen-like sequences andcarbohydrate recognition domains. The COLEC12 protein is a scavengerreceptor, a cell surface glycoprotein that displays several functionsassociated with host defense. The COLEC12 gene was suggested to be apossible biomarker candidate for anaplastic thyroid cancer(Espinal-Enriquez et al., 2015). COLEC12 was differentially expressed inHER2-positive breast cancer cell lines BT474 and might contribute totrastuzumab efficiency (von der Heyde et al., 2015).

Coatomer protein complex, subunit gamma 1 (COPG1)—COPG1 encodes aprotein subunit of the coatomer complex 1 (COPI). COPI-coated vesiclesmediate retrograde transport from the Golgi back to the ER andintra-Golgi transport. The cytosolic precursor of the COPI coat, theheptameric coatomer complex, can be thought of as composed of twosubcomplexes. The first consists of the beta-, gamma-, delta- andzeta-COP subunits which are distantly homologous to AP clathrin adaptorsubunits (Watson et al., 2004). EGF-dependent nuclear transport of EGFRis regulated by retrograde trafficking from the Golgi to the ERinvolving an association of EGFR with gamma-COP, one of the subunits ofthe COPI coatomer (Wang et al., 2010b). By immunohistochemisty, COPG1was confirmed to be abundantly expressed in lung cancer-derivedendothelial cells and in cancerous lung cells (Park et al., 2008).

CSNK2A2—Casein kinase II subunit alpha-prime is an enzyme that in humansis encoded by the CSNK2A2 gene. A retrospective study showed thatCSNK2A1 may be a useful prognosis marker in NSCLC patients aftercomplete resection, independent of lymph node metastasis status (Wang etal., 2010c). CSNK2A2 has been associated with tumor progression in latestage human colorectal cancer (Nibbe et al., 2009).

Dendrocyte expressed seven transmembrane protein (DCSTAMP)—This geneencodes a seven-pass transmembrane protein that is primarily expressedin dendritic cells. The encoded protein is involved in a range ofimmunological functions carried out by dendritic cells. DCSTAMP has beenidentified as a differentially expressed gene in papillary thyroidcarcinoma (Lee et al., 2009), and was subsequently confirmed to beexpressed at elevated levels in these samples (Kim et al., 2010).

Dyskeratosis congenita 1, dyskerin (DKC1)—The DKC1 gene functions in twodistinct complexes. Dyskerin mediates both the modification of uridineon ribosomal and small nuclear RNAs and the stabilization of thetelomerase RNA component (TERC). In human tumors dyskerin expression wasfound to be associated with both rRNA modification and TERC levels(Penzo et al., 2015). Moreover, dyskerin overexpression has been linkedto unfavorable prognosis in a variety of tumor types, e.g. in HCC (Liuet al., 2012).

Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2(DYRK2)/dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 4(DYRK4)—DYRK2 and DYRK4 belong to the family of Dyrk protein kinases(mammalian family with 5 members), which are involved in the regulationof cell differentiation, proliferation, and survival (Papadopoulos etal., 2011). DYRK2 controls the epithelial-mesenchymal transition inbreast cancer by degrading Snail (Mimoto et al., 2013). DYRK2 regulatesp53 to induce apoptosis and enhances the response to DNA damage: uponexposure to genotoxic stress, DYRK2 translocates into the nucleus andactivates p53 by phosphorylation (Meulmeester and Jochemsen, 2008; Tairaet al., 2007). The DYRK4 gene maps to chromosome 12p13.32, which wasdescribed as a susceptibility locus for CRC as the CCND2 gene isaffected (Jia et al., 2013; Peters et al., 2013). Some studies havehighlighted a role of DYRK4 in neuronal differentiation (Leypoldt etal., 2001; Slepak et al., 2012).

ERO1-like (S. cerevisiae) ERO1L—ERO1-like protein alpha is a proteinthat in humans is encoded by the ERO1L gene. ERO1-α is an oxidizingenzyme that exists in the endoplasmic reticulum and is induced underhypoxia. ERO1-α is overexpressed in a variety of tumor types. Moreover,the cancer-associated ERO1-α regulates the expression of the MHC class Imolecule via oxidative folding (Kukita et al., 2015). It has beensuggested that the expression of hERO1-α in cancer cells is associatedwith poorer prognosis and thus can be a prognostic factor for patientswith breast cancer (Kutomi et al., 2013). In natural human tumors, ERO1LmRNA was specifically induced in hypoxic microenvironments coincidingwith that of upregulated VEGF expression. It has been shown, thatreduction in ERO1L production via siRNA leads to significant inhibitionof VEGF secretion, a compromised proliferation capacity and enhancedapoptosis (May et al., 2005).

Family with sequence similarity 83, member A (FAM83A)—FAM83A wasdetermined to be elevated in several diverse cancer tissue types(Cipriano et al., 2014). However, the function of FAM83A remains unclear(Boyer et al., 2013). FAM83A was predicted a tumor-specific gene in lungcancer and its expression in lung cancer samples has been confirmedexperimentally. Expression was especially high in adenocarcinoma (Li etal., 2005). Others reported a correlation with lung cancer diseaseprogression (Liu et al., 2008).

Fragile X mental retardation, autosomal homolog 1 (FXR1)—The proteinencoded by the FXR1 gene is an RNA binding protein that interacts withthe functionally-similar proteins FMR1 and FXR2. FXR1 is deregulated ina variety of human disorders including cancer. FXR1 acted as an oncogenewhich could increase the proliferation, migration, and invasion ofcancer cells (Jin et al., 2015). FXR1 is a novel cancer gene in NSCLCand FXR1 executes its regulatory function by forming a novel complexwith two other oncogenes, protein kinase C, iota (PRKCI) and epithelialcell transforming 2 (ECT2) within the same amplicon in lung cancer cell(Qian et al., 2015b). It has been reported, that increased FXR1expression in NSCLC is a candidate biomarker predictive of poor survivaland might represent a novel therapeutic target. In addition, FXR1expression correlates with poor clinical outcome in multiple humancancers, suggesting broader implications of this RNA binding protein incancer progression (Qian et al., 2015a).

G2/M-phase specific E3 ubiquitin protein ligase (G2E3)—G2/Mphase-specific E3 ubiquitin-protein ligase is an enzyme that in humansis encoded by the G2E3 gene. G2E3 shuttles between the cytoplasm andnucleus, concentrating in nucleoli and relocalizing to the nucleoplasmin response to DNA damage. G2E3 is a dual function ubiquitin ligaseessential for prevention of apoptosis in early embryogenesis (Brooks etal., 2008). Some results suggest that G2E3 is a molecular determinant ofthe DNA damage response and cell survival, and that its loss sensitizestumor cells towards DNA-damaging treatment (Schmidt et al., 2015b).Moreover, loss of G2E3 triggered apoptosis and decreased proliferationof cancer cells. Thus, G2E3 acts as a survival factor (Schmidt et al.,2015a).

Guanylate binding protein 5 (GBPS)—The human guanylate binding protein 5(hGBP5) belongs to the family of interferon-gamma-inducible largeGTPases, which are well known for their high induction bypro-inflammatory cytokines (Wehner and Herrmann, 2010). hGBP5 exists inthree splice variants, forming two different proteins, of which thetumor-specific one is C-terminally truncated by 97 amino acids(Fellenberg et al., 2004).

Glutaminase (GLS)—The GLS gene encodes the K-type mitochondrialglutaminase. Glutaminase (GLS), which converts glutamine to glutamate,plays a key role in cancer cell metabolism, growth, and proliferation.Some studies demonstrate that GLS is required for tumorigenesis andsupport small molecule and genetic inhibition of GLS as potentialapproaches for targeting the tumor cell-autonomous dependence on GLS forcancer therapy (Xiang et al., 2015). Transient knock down of GLS splicevariants indicated that loss of GAC had the most detrimental effect onNSCLC cancer cell growth (van den Heuvel et al., 2012). The expressionof GLS1 is upregulated and correlates with clinicopathological factorsin colorectal cancer (Huang et al., 2014a), hepatocellular carcinoma(HCC) (Yu et al., 2015) and pancreatic ductal adenocarcinomas (PDA)(Chakrabarti et al., 2015).

Heat shock 70 kDa protein 2 (HSPA2)—HSPA2 has been identified as apotential cancer-promoting protein expressed at abnormal levels in asubset of human cancers, such as breast cancer (Mestiri et al., 2001),cervical cancer (Garg et al., 2010a), bladder urothelial cancer (Garg etal., 2010c), nasopharyngeal carcinoma (Jalbout et al., 2003) andmalignant tumors (Chouchane et al., 1997). Some level of the HSPA2 geneactivity was also observed in cell lines derived from several humancancers (Scieglinska et al., 2008), while silencing of the HSPA2 gene incancer cells led to growth arrest and decrease in tumorigenic potential(Rohde et al., 2005; Xia et al., 2008). Furthermore, polymorphism in theHSPA2 gene is associated with an increase in the risk of developing lungcancer (Wang et al., 2010a). Overexpression of HSPA2 is correlated withincreased cell proliferation, poor differentiation and lymph nodemetastases in human breast cancer, cervical cancer and bladderurothelial cancer (Garg et al., 2010a; Garg et al., 2010c; Mestiri etal., 2001).

Heat shock 70 kDa protein 8 (HSPA8)—The HSPA8 gene encodes a member ofthe heat shock protein 70 family Hsc70, which contains bothheat-inducible and constitutively expressed members. HSPA8 binds tonascent polypeptides to facilitate correct protein folding (Beckmann etal., 1990). Hsc70 function as molecular chaperones, assisting in proteinsynthesis, folding, assembly, trafficking between cellular compartments,and degradation (Bukau and Horwich, 1998; Hartl and Hayer-Hartl, 2002).Hsc70 is expressed in non-malignant mammary cells as well as breastcancer cells (Kao et al., 2003; Vargas-Roig et al., 1998) and theoverexpression of Hsp/hsc70 in chemoresistant cancer cells (Ciocca etal., 1992; Lazaris et al., 1997) has prompted studies about possibleclinical markers of these proteins (Ciocca and Calderwood, 2005). Thereis a potential role of this secreted hsc70 chaperone in cellproliferation that might account for the higher tumor growth of cancercells overexpressing cathepsin D (Nirde et al., 2010). FurthermoreRuisin et al. reported an association between a polymorphism of thisgene and lung cancer risk (Rusin et al., 2004).

Heat shock 70 kDa protein 1A (HSPA1A)—HSPA1A, also known as HSP72, wasshown to be strongly upregulated in cancers and to play a critical rolefor tumor cell growth by suppressing p53-dependent and p53-independentsenescence pathways (Sherman, 2010). Overexpression is described for RCC(Atkins et al., 2005) and gastrointestinal carcinomas (Wang et al.,2013a), the latter showing a significant correlation with progression,infiltration and the presence of lymph node and remote metastasis.

Heat shock 70 kDa protein 1B (HSPA1B)—HSPA1B, also known as HSP70-2,encodes the testis specific heat-shock protein 70-2, essential for thegrowth of spermatocytes and cancer cells (Hatfield and Lovas, 2012).Different studies suggest an important role of HSP70-2 in diseaseprogression of cervical cancer (Garg et al., 2010b), renal cellcarcinoma (Singh and Suri, 2014) and bladder cancer and polymorphismswithin the gene are associated with the development of gastric cancer(Ferrer-Ferrer et al., 2013). Some functional HSPA1B variants areassociated with lung cancer risk and survival. These Hsp70 geneticvariants may offer useful biomarkers to predict lung cancer risk andprognosis (Szondy et al., 2012; Guo et al., 2011).

Heat shock 70 kDa protein 1-like (HSPA1L)—Heat shock 70 kDa protein 1Lis a protein that in humans is encoded by the HSPA1L gene on chromosome6. Though it shares close homology to HSPA1A and HSPA1B, it is regulateddifferently and is not heat-inducible (Ito et al., 1998). Polymorphismswithin the gene are associated with prostate cancer susceptibility andprognosis (Sfar et al., 2010) and with hepatocellular carcinomasusceptibility (Medhi et al., 2013).

Heat shock 70 kDa protein 6 (HSP70B′) (HSPA6)—Heat shock protein (Hsp)70B′ is a human Hsp70 chaperone that is strictly inducible, havinglittle or no basal expression levels in most cells (Noonan et al.,2007). HSPA6, also known as heat shock protein 70B′, was shown to beupregulated by Y15 treatment in glioblastoma cells (Huang et al., 2014b)and heat shock in head and neck cancer cells (Narita et al., 2002). Highlevels of HSPA6 might be associated with earlier recurrence of HCC (Yanget al., 2015).

Heat shock 70 kDa protein 7 (HSP70B) (HSPA7)—HSPA7 is a pseudogene.

HSPA (heat shock 70 kDa) binding protein, cytoplasmic cochaperone 1(HSPBP1)—Heat shock-binding protein HspBP1 is a member of the Hsp70co-chaperone family. HspBP1 is a co-chaperone that binds to andregulates the chaperone Hsp70. The levels of HspBP1 and Hsp70 weresignificantly higher in sera of breast cancer patients compared to seraof healthy individuals (Souza et al., 2009). HSPBP1 was over-expressedin patients with leukemia (Sedlackova et al., 2011). HspBP1 wasup-regulated in human HCV-HCC, an increase which correlated with theincrease of Hsp70 levels (Yokoyama et al., 2008).

IQ motif containing GTPase activating protein 1 (IQGAP1)—IQGAP1, alsoknown as p195, is a ubiquitously expressed protein that in humans isencoded by the IQGAP1 gene. IQGAP1 is a key mediator of several distinctcellular processes, in particular cytoskeletal rearrangements. Recentstudies have implicated a potential role for IQGAP1 in cancer, supportedby the over-expression and distinct membrane localization of IQGAP1observed in a range of tumors (Johnson et al., 2009). Theover-expression of IQGAP1 may play an important role in pancreaticcancer occurrence and progression (Wang et al., 2013c). SuppressingIQGAP1 expression reduced the tumor cell growth, migration and invasionin esophageal squamous cell carcinoma (ESCC) (Wang et al., 2014c).Furthermore, increased IQGAP1 expression during the differentiation ofovarian cancer stem cell-like cells (CSC-LCs) is involved in anaggressive cell behavior, which may contribute to metastasis of ovariancancer (Huang et al., 2015a).

Integrin, beta 6 (ITGB6)—ITGB6 is a subtype of integrin that isexpressed exclusively on the surfaces of epithelial cells and is areceptor for extracellular matrix proteins (Weinacker et al., 1994). Astudy found increased expression of ITGB6 in 10 human tumor typesstudied relative to normal tissues. Highest frequency of ITGB6expression was reported for squamous carcinomas of the cervix, skin,esophagus, and head and neck. Of note, antibody-mediated blockade ofITGB6 inhibited tumor progression in vivo (Van Aarsen et al., 2008).ITGB6 has been exploited as target for tumor-specific drug delivery andenhanced therapeutic efficacy in colon carcinoma (Liang et al., 2015;Zhao-Yang et al., 2008). In breast cancer high expression of either themRNA or protein for ITGB6 was associated with very poor survival andincreased metastases to distant sites. An antibody targeting ITGB6inhibited tumor growth in breast cancer mouse models (Allen et al.,2014).

Lysine (K)-specific demethylase 6B (KDM6B)—KDM6B, also known as JMJD3,is a histone demethylase that in humans is encoded by the KDM6B gene.KDM6B affects transcriptional regulation by demethylating lysine 27residue of histone 3. Low KDM6B expression was an independent predictorof poor prognosis (P=0.042) in surgically resected CRC patients(Yokoyama et al., 2008). Moreover, over-expression of KDM6B inhibitedcell growth by initiating mitochondria-dependent apoptosis and byattenuating the invasion-metastasis cascade in NSCLC cells (Ma et al.,2015). On the other hand, KDM6B has high expression level in clear cellrenal cell carcinoma (ccRCC) and is positively correlated with poorccRCC prognosis. Knockdown of KDM6B could inhibit ccRCC tumorigenesis invitro (Li et al., 2015). Furthermore, deregulation of KDM6B maycontribute to gliomagenesis via inhibition of the p53 pathway resultingin a block to terminal differentiation (Ene et al., 2012).

Keratin 9, type I (KRT9)—Keratin 9 is a type I cytokeratin that inhumans is encoded by the KRT9 gene. It is found only in the terminallydifferentiated epidermis of palms and soles. Mutations in the geneencoding this protein cause epidermolytic palmoplantar keratoderma (Reiset al., 1994). KRT9 was up-regulated in HCC. This over-expression mayplay a crucial role in HCC metastasis, and can be used as a potentialserum marker for predicting HCC metastasis (Fu et al., 2009).

LINE1 retrotransposable element 1 (L1RE1)—The L1RE1 gene, also known asLRE1, encodes a ‘LINE’ (long interspersed nuclear element)retrotranposable element (LRE), a mobile DNA sequence with autonomousretrotransposon activity. The family of LINE1 retrotransposons isreportedly hypomethylated in many cancers and reflects globalmethylation status in the genome (Ostertag and Kazazian, Jr., 2001). Onelong interspersed nuclear element repeat region, LRE1, located on22q11-q12, is a consistent indicator of global methylation status(Chalitchagorn et al., 2004; Ostertag and Kazazian, Jr., 2001). Somedata suggest that LRE1 relative methylation is an independent epigeneticbiomarker of head and neck squamous cell carcinoma (HNSCC) (Hsiung etal., 2007).

Laminin, beta 3 (LAMB3)—LAMB3 encodes the beta 3 subunit of laminin,which together with an alpha and a gamma subunit, forms laminin-5. LAMB3was up-regulated in papillary thyroid carcinoma (PTC) (Barros-Filho etal., 2015), cervical squamous cell carcinoma (cervical SCC) (Yamamoto etal., 2013) and oral squamous cell carcinoma (OSCC) (Tanis et al., 2014).Gene array and bioinformatics analyses implied that LAMB3 was a key geneinvolved in lung cancer. Knockdown of this gene suppressed human lungcancer cell invasion and metastasis in vitro and in vivo. LAMB3 wasover-expressed in lung cancer patients and its expression correlatedwith lymphatic metastasis (Wang et al., 2013b).

Lysosomal protein transmembrane 5 (LAPTMS)—The LAPTMS gene encodes amembrane protein on the intracellular vesicles that is associated withlysosomes. LAPTMS is aberrantly methylated in lung cancer, and themethylation was correlated with the differentiation state of the tumor(Cortese et al., 2008). The accumulation of LAPTMS-positive vesicles wasclosely associated with the programmed cell death occurring during thespontaneous regression of neuroblastomas (Inoue et al., 2009). The CD1eprotein participates in the presentation of lipid antigens in dendriticcells. LATPMS controls either CD1e ubiquitination or the generation ofsoluble lysosomal CD1e proteins (Angenieux et al., 2012).

Minichromosome maintenance complex component 4 (MCM4)—The proteinencoded by the MCM4 gene is one of the highly conserved mini-chromosomemaintenance proteins (MCM) that are essential for the initiation ofeukaryotic genome replication. MCM4 was down-regulated in bladder cancer(Zekri et al., 2015) and differentially expressed in lung adenocarcinomain comparison with normal lung tissues (Zhang et al., 2014). MCM4over-expression was associated with shorter survival in breast cancerpatients (Kwok et al., 2015).

Minichromosome maintenance complex component 5 (MCM5)—MCM5 is implicatedin DNA replication and cell cycle regulation. High expression levels ofMCM5 were shown to be associated with progression and poorer prognosisin oral squamous cell carcinoma (Yu et al., 2014), cervical cancer (Daset al., 2013), gastric cancer (Giaginis et al., 2011) and colon cancer(Burger, 2009).

Melanoregulin (MREG)—MREG plays a role in intracellular melanosomedistribution (Wu et al., 2012), though regulation of retrogrademicrotubule-dependent melanosome transport (Ohbayashi et al., 2012).Moreover, MREG also functions in regulation of pigment incorporationinto melanosomes (Rachel et al., 2012). MREG was shown to be targeted bymiRNA-26 in its 3′ UTR in estrogen receptor-positive breast cancercells. However, a direct involvement of MREG in miRNA-26 mediated cellproliferation could not be demonstrated (Tan et al., 2014).

NODAL modulator 1 (NOMO1)/NODAL modulator 2 (NOMO2)/NODAL modulator 3(NOMO3)—The NOMO1, NOMO2 and NOMO3 genes are three highly similar genesin a region of duplication located on the p arm of chromosome 16. Thesethree genes encode closely related proteins that may have the samefunction. NOMO1 was identified as over-expressed gene in cutaneousT-cell lymphoma (CTCL) cell line HuT78 (Lange et al., 2009). NOMO1 is anantagonist of Nodal signaling. Nodals are signaling factors of thetransforming growth factor-beta (TGFbeta) superfamily with a key role invertebrate development (Haffner et al., 2004).

Nucleoporin 153 kDa (NUP153)—Nucleoporin 153 (Nup153), a component ofthe nuclear pore complex (NPC), has been implicated in the interactionof the NPC with the nuclear lamina. Nup153 depletion induces a dramaticcytoskeletal rearrangement that impairs cell migration in human breastcarcinoma cells (Zhou and Pante, 2010). The NUP153 nucleoporin regulatesthe distribution of specific proteins between the nucleus and thecytoplasm, interestingly including the transducer of TGFβ signaling,SMAD2 (Xu et al., 2002). Recently, some analysis revealed novel possibleoncogenic functions of nucleoporin NUP153 (ostensibly by modulating TGFβsignaling) in pancreatic cancer (Shain et al., 2013).

PERP, TP53 apoptosis effector (PERP)—PERP is a p53/p63-regulated geneencoding a desmosomal protein that plays a critical role in cell-celladhesion and tumor suppression. Loss of PERP expression correlates withthe transition to squamous cell carcinoma (SCC) and with increased localrelapse in patients with oral cavity SCC (Kong et al., 2013). PERPexpression was reduced in many human breast cancer cell lines (Dusek etal., 2012). Some studies suggested that Perp-deficiency promoted cancerby enhancing cell survival, desmosome loss, and inflammation (Beaudry etal., 2010). PERP is an apoptosis-associated target of p53, and itsactivation alone is sufficient to induce apoptotic pathway leading tocell death (Chen et al., 2011).

Putative homeodomain transcription factor 1 (PHTF1)/Putative homeodomaintranscription factor 2 (PHTF2)—PHTF1 (putative homeodomaintranscriptional factor) is a putative homeobox gene located at 1p11-p13in the human genome. This gene is evolutionarily conserved and mainlyexpressed in the testis (Manuel et al., 2000). As a transcriptionfactor, the PHTF1 gene is mainly involved in biological processes suchas DNA-dependent transcription and the regulation of biologicalprocesses. PHTF1 over-expression is responsible for regulating cellproliferation and apoptosis in T cell acute lymphoblastic leukemia(T-ALL) cell lines. PHTF1 may be a tumor-suppressor like gene and atherapeutic target for triggering the PHTF1-FEM1b-Apaf-1 apoptosispathway (Huang et al., 2015b).

Putative homeodomain transcription factor 2 is a protein that in humansis encoded by the PHTF2 gene. PHTF2 is predominantly expressed in muscleand is located at 7q11.23-q21 in the human genome (Manuel et al., 2000).

Pleckstrin homology domain containing, family M (with RUN domain) member1 (PLEKHM1)—The protein encoded by the PLEKM1 gene is essential for boneresorption, and may play a critical role in vesicular transport in theosteoclast. Mutations in this gene are associated with autosomalrecessive osteopetrosis type 6 (OPTB6) (van et al., 2004). PLEKHM1 wassuggested to be a candidate susceptibility gene for epithelial ovariancancer (Permuth-Wey et al., 2013).

Phospholipid transfer protein (PLTP)—Phospholipid transfer protein(PLTP) plays an important role in regulation of inflammation. Some datasuggest that PLTP has anti-inflammatory capabilities in macrophages(Vuletic et al., 2011). Moreover, PLTP is essential in mediating theassociation of triacyl lipid A with lipoproteins, leading to extensionof its residence time and to magnification of its proinflammatory andanticancer properties (Gautier et al., 2010). PLTP was differentiallyexpressed in breast cancer patient and might be associated withchemotherapy response (Chen et al., 2012).

Protein phosphatase 2, regulatory subunit B″, alpha (PPP2R3A)—This geneencodes one of the regulatory subunits of the protein phosphatase 2.Protein phosphatase 2 (formerly named type 2A) is one of the four majorSer/Thr phosphatases and is implicated in the negative control of cellgrowth and division (Ruediger et al., 2001). PPP2R3A was frequentlymethylated in childhood acute lymphoblastic leukemia (ALL) (Dunwell etal., 2009).

PTC7 protein phosphatase homolog (S. cerevisiae) (PPTC7)—PPTC7 encodesPTC7 protein phosphatase homolog and is located on chromosome 12q24.11.PPTC7 was recently identified as novel susceptibility gene in responseto environmental toxicants (Zhu et al., 2015).

Protein kinase, DNA-activated, catalytic polypeptide (PRKDC)—PRKDCencodes the catalytic subunit of the DNA-dependent protein kinase(DNA-PK), a member of the PI3/P14-kinase family. It was shown that PRKDCmay stabilize the c-Myc oncoprotein via Akt/GSK3 pathway (An et al.,2008). Activation of PRKDC positively correlated with HCC proliferation,genomic instability and microvessel density, and negatively withapoptosis and patient's survival (Evert et al., 2013).

Proteasome (prosome, macropain) subunit, alpha type, 4 (PSMA4)—PSMA4encodes proteasome subunit alpha 4, which cleaves peptides in anATP/ubiquitin-dependent process in a non-lysosomal pathway. Singlenucleotide polymorphisms in the PSMA4 gene have been associated with therisk of lung cancer in Chinese Han population (Wang et al., 2015). Onthe other side, it has been reported that single nucleotidepolymorphisms in the PSMA4 gene are not major contributors to non-smallcell lung cancer susceptibility (Yongjun Zhang et al., 2013).Furthermore, over-expression of PSMA4 was observed in lung tumorscompared with normal lung tissues. Down-regulation of PSMA4 expressiondecreased proteasome activity and induced apoptosis (Liu et al., 2009).

Protein tyrosine phosphatase, non-receptor type 13 (PTPN13)—This geneencodes a member of the protein tyrosine phosphatase (PTP) family. PTPsare signaling molecules that regulate a variety of cellular processesincluding cell growth, differentiation, mitotic cycle and oncogenictransformation. PTPN13 was found to interact with the Fas receptor andmight therefore have a role in Fas mediated programmed cell death.Moreover, PTPN13 interacts with GTPase-activating protein and thus mayfunction as a regulator of Rho signaling pathways. In hematologicalmalignancies PTPN13 has contradictory effects, either suppressing orpromoting tumor growth, in lymphoma and in myeloid leukemia,respectively (Wang et al., 2014b). This can be explained by the capacityof PTPN13 to counteract the activity of oncogenic tyrosine kinases andits inhibitory interaction with the Fas death receptor (Freiss andChalbos, 2011). In breast cancer, PTPN13 was regarded as a unique markerof mammary tumor response to antiestrogens and a potential therapeutictarget to activate apoptotic stimuli in tumor cells (Freiss et al.,2004). The inhibition of the Fas/PTPN13 binding might provide a goodtarget to develop anti-cancer drugs (Takahashi and Kataoka, 1997).

RAS p21 protein activator 2 (RASA2)—RAS p21 protein activator 2 encodesa member of the GAP1 family of GTPase-activating proteins. Acting as asuppressor of RAS function, RASA2 enhances the weak intrinsic GTPaseactivity of RAS proteins resulting in the inactive GDP-bound form ofRAS, thereby allowing control of cellular proliferation anddifferentiation. Depending on the precise genetic alteration, itslocation within the gene and the effects it exerts on protein function,RASA2 can theoretically function as either an oncogene or as a tumorsuppressor gene (Friedman, 1995). Under mild stress conditions, RASA2 iscleaved by caspase-3 which results in a fragment called fragment Nstimulating anti-death signaling. When caspase-3 activity furtherincreases, this generates a fragment, called N2, which no longerprotects cells. On the other hand, full-length RASA2 favors Akt activityby shielding it from deactivating phosphatases (Cailliau et al., 2015).In breast cancer, stress-activated caspase-3 might contribute to thesuppression of metastasis through the generation of fragment N2 (Barraset al., 2014). RASA2 was identified as a tumor-suppressor gene mutatedin 5% of melanomas (Arafeh et al., 2015).

Recombination signal binding protein for immunoglobulin kappa J region(RBPJ)—Recombination signal binding protein for immunoglobulin kappa Jregion encodes a transcriptional regulator important in the Notchsignaling pathway. RBPJ acts as a repressor when not bound to Notchproteins and an activator when bound to Notch proteins. It is thought tofunction by recruiting chromatin remodeling complexes containing histonedeacetylase or histone acetylase proteins to Notch signaling pathwaygenes. Xenograft mouse models showed that RBPJ knockdown inhibitedtumorigenicity and decreased tumor volume suggesting that hypoxiapromotes Smoothened transcription through up-regulation of RBPJ toinduce proliferation, invasiveness and tumorigenesis in pancreaticcancer (Onishi et al., 2016). The effect that RBPJ knockdown led to asignificant decrease in cell growth was also found in prostate and lungcancer cells, suggesting that RBPJ expression could be a promisingtherapeutic approach for treating human cancer (Xue et al., 2015; Lv etal., 2015). Moreover, over-expression of RBPJ promoted theanchorage-independent growth of rhabdomyosarcoma cells (Nagao et al.,2012). The RBPJ-mediated Notch signaling is also essential for dendriticcell-dependent anti-tumor immune responses (Feng et al., 2010).

Sterile alpha motif domain containing 9-like (SAMD9L)—SAMD9L encodessterile alpha motif domain containing 9-like and is located onchromosome 7q21.2. SAMD9 and SAMD9L genes share a common gene structureand encode proteins with 60% amino acid identity with a suggested rolein suppressing inflammatory pathways. SAMD9L localizes in earlyendosomes and acts as an endosome fusion facilitator. Haploinsufficiencyof SAMD9L gene contributes to myeloid transformation and

SAMD9L was identified as candidate myeloid tumor suppressor gene(Nagamachi et al., 2013). SAMD9L knockdown significantly promoted cellproliferation and colony formation of hepatocellular carcinoma celllines as SAMD9L silence facilitated G1-S transition of cell cycleprogression and led to the elevated activity of Wnt/beta-cateninpathway. Recent findings highlight the tumor-suppressive role of SAMD9Linactivation by somatic mutation and decreased expression in humancancers (Wang et al., 2014a). SAMD9L exhibited significantly decreasedexpression in T and B cell populations of patients with metastaticmelanoma as compared with those from healthy control individuals(Critchley-Thorne et al., 2007).

Splicing factor 3b, subunit 3, 130 kDa (SF3B3)—SF3B3 encodes subunit 3of the splicing factor 3b protein complex. Over-expression of SF3B3 issignificantly correlated with overall survival and endocrine resistancein estrogen receptor-positive breast cancer (Gokmen-Polar et al., 2015).

Surfactant protein A1 (SFTPA1)/Surfactant protein A2 (SFTPA2)—Thesegenes encode lung surfactant proteins that are a member of a subfamilyof C-type lectins called collectins. SFTPAs bind specific carbohydratemoieties found on lipids and on the surface of microorganisms and playan essential role in surfactant homeostasis and in the defense againstrespiratory pathogens. Mutations in these genes are associated withidiopathic pulmonary fibrosis. A lung cancer-specific gene signature,containing SFTPA1 and SFTPA2 genes, accurately distinguished lung cancerfrom other cancer samples (Peng et al., 2015). EGFR mutations weresignificantly more common in pulmonary adenocarcinoma with SFTPAexpressions than in those without (Jie et al., 2014). SFTPA suppresseslung cancer progression by regulating the polarization oftumor-associated macrophages (Mitsuhashi et al., 2013). Expression ofmutant SFTPA2 in lung epithelial cells leads to secretion of latentTGF-beta1 and TGF-beta1 mediated EMT (Maitra et al., 2012). Moreover,the development of prostate cancer may be related to decreased level ofSFTPA (Kankavi et al., 2014).

Solute carrier family 25 (mitochondrial carrier; adenine nucleotidetranslocator), member 31 (SLC25A31)/solute carrier family 25(mitochondrial carrier; adenine nucleotide translocator), member 4(SLC25A4)/solute carrier family 25 (mitochondrial carrier; adeninenucleotide translocator), member 5 (SLC25A5)/solute carrier family 25(mitochondrial carrier; adenine nucleotide translocator), member 6(SLC25A6)—Proteins of the solute carrier family 25 are ADP/ATP carrierthat exchange cytosolic ADP for matrix ATP in the mitochondria. Theyfunction as a gated pore that translocates ADP/ATP and form a homodimerembedded in the inner mitochondria membrane. Cells over-expressing thisgene family have been shown to display an anti-apoptotic phenotype.Suppressed expression of this gene family has been shown to induceapoptosis and inhibit tumor growth. While SLC25A4 is preferentiallypresent in differentiated tissues and is specific for muscle and brain,SLC25A5 is expressed in proliferating tissues such as tumors. SLC25A6 isexpressed ubiquitously and SLC25A31 is present in liver and germ cells(Dolce et al., 2005). Especially SLC25A5 contributes to carcinogenesis.Since the expression of SLC25A5 is closely linked to the mitochondrialbioenergetics of tumors, it is a promising target for individualizingcancer treatments and for the development of anticancer strategies(Chevrollier et al., 2011). Moreover, stable over-expression of SLC25A31protected cancer cells from ionidamine and staurosporine apoptosisindependent of Bcl-2 expression. Therefore, dichotomy is found in thehuman SLC25 isoform sub-family with SLC25A4 and SLC25A6 isoformsfunctioning as pro-apoptotic, while SLC25A5 and SLC25A31 isoforms rendercells resistant to death inducing stimuli (Gallerne et al., 2010).

SP140 nuclear body protein (SP140)—SP140 encodes the SP140 nuclear bodyprotein and is located on chromosome 2q37.1. SP140 was shown to beup-regulated in laryngeal squamous cell carcinoma (Zhou et al., 2007).SP140 is associated with chronic lymphocytic leukemia (Lan et al.,2010), multiple myeloma (Kortum et al., 2015) and acute promyelocyticleukemia (Bloch et al., 1996).

Signal transducer and activator of transcription 1, 91 kDa (STAT1)—STAT1is activated by tyrosine phosphorylation in response to all interferons(Decker et al., 2002) and contributes to Th1 cell differentiation(Schulz et al., 2009). At the molecular level, STAT1 inhibits theproliferation of both mouse and human tumor cells treated with IFN-γ viaits ability to increase the expression of cyclin-dependent kinaseinhibitor p21Cip1, or to decrease c-myc expression (Ramana et al.,2000). The anti-tumor activity of STAT1 is further supported by itsability to inhibit angiogenesis and tumor metastasis in mouse models(Huang et al., 2002). Increased STAT1 mRNA levels were shown to be partof a molecular signature associated with better prediction of themetastatic outcome for patients with hormone receptor negative andtriple-negative breast cancers (Yau et al., 2010).

Transmembrane protein 43 (TMEM43)—This gene encodes transmembraneprotein 43. Defects in this gene are the cause of familialarrhythmogenic right ventricular dysplasia type 5 (ARVD5), also known asarrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). ARVD isan inherited disorder and is characterized by ventricular tachycardia,heart failure, sudden cardiac death and fibrofatty replacement ofcardiomyocytes (Siragam et al., 2014). TMEM43 may have an important rolein maintaining nuclear envelope structure by organizing proteincomplexes at the inner nuclear membrane (Bengtsson and Otto, 2008).

Topoisomerase (DNA) II alpha 170 kDa (TOP2A)/topoisomerase (DNA) II beta180 kDa (TOP2B)—TOP2A and TOP2B encode highly homologous isoforms of aDNA topoisomerase, an enzyme that controls and alters the topologicstates of DNA during transcription. This nuclear enzyme is involved inprocesses such as chromosome condensation, chromatid separation, and therelief of torsional stress that occurs during DNA transcription andreplication. TOP2A is essential for cell proliferation and is highlyexpressed in vigorously growing cells, whereas TOP2B is nonessential forgrowth and has recently been implicated in treatment-associatedsecondary malignancies (Toyoda et al., 2008). TOP2A has found to beover-expressed in several cancer types (e.g. malignant pleuralmesothelioma (Roe et al., 2010), malignant peripheral nerve sheath tumor(Kresse et al., 2008), lung adenocarcinoma cells (Kobayashi et al.,2004), bladder cancer (Simon et al., 2003), glioblastomas (van den Boomet al., 2003)). TOP2B is involved in DNA transcription, replication,recombination, and mitosis, and besides TOP1, represents the secondNUP98 fusion partner gene that belongs to the topoisomerase gene family(Nebral et al., 2005).

Tryptase alpha/beta 1 (TPSAB1)/tryptase beta 2 (TPSB2)—Tryptasealpha/beta 1 (TPSAB1) and tryptase beta 2 (TPSB2) are together with twoother tryptase isoforms, expressed by mast cells. Tryptases have beenimplicated as mediators in the pathogenesis of asthma and other allergicand inflammatory disorders. Tryptase secreted by mast cells haspro-angiogenic function and contributes to tumor vascularization.Tryptase acts by activation of protease-activated receptor-2 (PAR-2) andadditionally contributes to extracellular matrix degradation, thus alsofacilitating vessel growth. Moreover, the presence of tryptase-positivemast cells in tumor tissue correlates with angiogenesis in severalcancer types (Ammendola et al., 2014). Elevated levels oftryptase-positive mast cells have been reported in prostate cancer andhave been correlated with microvessel density, tumor stage, and shortersurvival (Nonomura et al., 2007; Stawerski et al., 2013). Similarly,tryptase-positive mast cells are also associated with tumor stage andangiogenesis in gastric cancer (Zhao et al., 2012; Ribatti et al., 2010)as well as in lung adenocarcinoma (Imada et al., 2000; Takanami et al.,2000).

Tripartite motif containing 11 (TRIM11)—Tripartite motif-containingprotein 11 is a protein that in humans is encoded by the TRIM11 gene.TRIM11 is known to be involved in the development of the central nervoussystem and to destabilize humanin, an inhibitor of Alzheimer-likeneuronal insults (Niikura et al., 2003). TRIM11 is overexpressed inhigh-grade gliomas and promotes proliferation, invasion, migration andglial tumor growth (Di et al., 2013).

Transient receptor potential cation channel, subfamily M, member 2(TRPM2)—The protein encoded by this gene is a calcium-permeable cationchannel that is regulated by free intracellular ADP-ribose. TRPM2 mightbe involved in mediating apoptosis under certain conditions (Ishii etal., 2007; Cao et al., 2015). However, its effect on cell growthproliferation is less clear and might depend on cell culture conditionsand the expression of alternatively spliced isoforms (Chen et al.,2014). In melanoma and prostate cancer, a tumor-enriched TRPM2 antisensetranscript has been identified which is correlated with apoptosis andclinical outcome (Orfanelli et al., 2015).

Tubulin gamma complex associated protein 3 (TUBGCP3)—Tubulin gammacomplex associated protein 3 is part of the multi-subunit gamma-tubulincomplex that is critical for microtubule nucleation in eukaryotic cells(Lynch et al., 2014). Cytoplasmic gamma-tubulin complexes are targetedto centrosomes or to other microtubule organizing centers via a set ofso called gamma-tubulin complex binding proteins (Schiebel, 2000). Asignificant increase in the expression of TUBGCP3 transcripts inglioblastoma cells versus normal human astrocytes was found and TUBGCP3immunoreactivity was significantly increased over that in normal brains.TUBGCP3 was also associated with microvascular proliferation andinteraction with signaling pathways leading to a malignant phenotype(Draberova et al., 2015). Moreover, TUBGCP3 was found to besignificantly higher expressed in near-tetraploid than in diploid mantlecell lymphoma samples (Neben et al., 2007).

Ubiquitin-like modifier activating enzyme 6 (UBA6)—Ubiquitin-likemodifier-activating enzyme 6 is a protein that in humans is encoded bythe UBA6 gene. UBA6 is an ubiquitin-activating enzyme being mostabundantly expressed in the testis. Further it is required for cellularresponse to DNA damage (Moudry et al., 2012).

Xenotropic and polytropic retrovirus receptor 1 (XPR1)—XPR1 is amultipass membrane molecule that contains a 180-residue-longaminoterminal SPX domain (named after SYG1, Pho81, and XPR1). XPR1 hasbeen reported to mediate phosphate export (Giovannini et al., 2013).Upon osteoclast differentiation XPR1 mRNA transcripts were found toincrease (Sharma et al., 2010). Originally, XPR1 was described asretroviral receptor, used by xenotropic and polytropic MLV (X-MLV andP-MLV) two gammaretroviruses that can infect human cells as well asvarious other species, such as mice and birds (Kozak, 2010; Martin etal., 2013).

Zinc finger BED-type containing 5 (ZBED5)—Zinc finger BED-typecontaining 5 is characterized by a coding sequence that is mostlyderived from Charlie-like DNA transposon, however, it does not appear tobe an active DNA transposon as it is not flanked by terminal invertedrepeats. ZBED5 is related to Buster DNA transposons and isphylogenetically separate from other ZBED5. ZBED genes are widelyexpressed among vertebrate tissues and together they regulate aremarkable diversity of functions (Hayward et al., 2013).

Zinc finger protein 697 (ZNF697)—The ZNF697 gene encodes zinc fingerprotein 697 that is located on chromosome 1p12 and probably plays a rolein DNA binding (Yu et al., 2011).

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, or 13 or longer, andin case of MHC class II peptides (elongated variants of the peptides ofthe invention) they can be as long as 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 11 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mod et al.(Mod et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype from Allele Populationallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7% DR9 Caucasian (North America)   2.1% DR1 African (North) American13.20% DR2 African (North) American 29.80% DR3 African (North) American24.80% DR4 African (North) American 11.10% DR5 African (North) American31.10% DR6 African (North) American 33.70% DR7 African (North) American19.20% DR8 African (North) American 12.10% DR9 African (North) American 5.80% DR1 Asian (North) American  6.80% DR2 Asian (North) American33.80% DR3 Asian (North) American  9.20% DR4 Asian (North) American28.60% DR5 Asian (North) American 30.00% DR6 Asian (North) American25.10% DR7 Asian (North) American 13.40% DR8 Asian (North) American12.70% DR9 Asian (North) American 18.60% DR1 Latin (North) American15.30% DR2 Latin (North) American 21.20% DR3 Latin (North) American15.20% DR4 Latin (North) American 36.80% DR5 Latin (North) American20.00% DR6 Latin (North) American 31.10% DR7 Latin (North) American20.20% DR8 Latin (North) American 18.60% DR9 Latin (North) American 2.10% A*24 Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan  59% A*24 Malaysia   58% A*24:02 Philippines   54% A*24 India   47%A*24 South Korea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02India   29% A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara  20% A*24 South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to HLA-A*02 and HLA-A*24. TheMHC class II peptides of the invention bind to several different HLAclass II molecules and are called promiscuous binders (pan-bindingpeptides). A vaccine may also include pan-binding MHC class II peptides.Therefore, the vaccine of the invention can be used to treat cancer inpatients that are A*02 or A*24 positive, whereas no selection for MHCclass II allotypes is necessary due to the pan-binding nature of thesepeptides.

If A*02 peptides of the invention are combined with A*24 peptides of theinvention, a higher percentage of any patient population can be treatedcompared with addressing either MHC class I allele alone. While in mostpopulations less than 50% of patients could be addressed by eitherallele alone, a vaccine comprising HLA-A*24 and HLA-A*02 epitopes cantreat at least 60% of patients in any relevant population. Specifically,the following percentages of patients will be positive for at least oneof these alleles in various regions: USA 61%, Western Europe 62%, China75%, South Korea 77%, Japan 86% (calculated fromwww.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences; and R is the number of bases or amino acids in the ReferenceSequence over the length of the alignment with the Compared Sequencewith any gap created in the Reference Sequence also being counted as abase or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 110 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 110, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 110. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 110, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 12 Preferred variants and motif of the HLA-A*02 peptides according to SEQ ID NO: 1, 2, and 4 Position 1 2 3 4 5 6 7 8 9SEQ ID 1 K L L P Y I V G V Variant I L A M M I M L M A A A I A L A A V VI V L V A T T I T L T A Q Q I Q L Q A Position 1 2 3 4 5 6 7 8 9SEQ ID 2 F L I P Y A I M L Variant V I A M V M I M M A A V A I A A A V VV I V V A T V T I T T A Q V Q I Q Q A Position 1 2 3 4 5 6 7 8 9SEQ ID 4 F V F S F P V S V Variant L L I L L L A M M I M L M A A A I A LA A I L A T T I T L T A Q Q I Q L Q A

TABLE 12B Preferred variants and motif of the HLA-A*02 peptide according to SEQ ID NO: 13 Position 1 2 3 4 5 6 7 8 9 SEQ ID 13F L F D G S A N L Variant V I A M V M I M M A A V A I A A A V V V I V VA T V T I T T A Q V Q I Q Q A

TABLE 13 Preferred variants and motif of the HLA-A*24 peptides according to SEQ ID NO: 23, 24, and 25 Position 1 2 3 4 5 6 78 9 10 11 SEQ ID 23 V Y T S W Q I P Q K F Variant I F L F I F L Position1 2 3 4 5 6 7 8 9 10 11 SEQ ID 24 N Y P K S I H S F Variant I L F I F LF Position 1 2 3 4 5 6 7 8 9 10 11 SEQ ID 25 R F M D G H I T F Variant YI Y L Y I L

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 14.

TABLE 14 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 μM, andmost preferably no more than about 10 μM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 110.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 110 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich(http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other alpha-amino groups is, for example, useful in bindingof peptides to surfaces or the cross-linking of proteins/peptides.Lysine is the site of attachment of poly(ethylene)glycol and the majorsite of modification in the glycosylation of proteins. Methionineresidues in proteins can be modified with e.g. iodoacetamide,bromoethylamine, and chloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004) and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESl) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of natural TUMAPsrecorded from lung cancer (NSCLC) samples (N=91 A*02-positive samplesand N=80 A*24-positive samples) with the fragmentation patterns ofcorresponding synthetic reference peptides of identical sequences. Sincethe peptides were directly identified as ligands of HLA molecules ofprimary tumors, these results provide direct evidence for the naturalprocessing and presentation of the identified peptides on primary cancertissue obtained from 155 lung cancer (NSCLC) patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from lung cancer (NSCLC) tissue samples werepurified and HLA-associated peptides were isolated and analyzed by LC-MS(see examples). All TUMAPs contained in the present application wereidentified with this approach on primary lung cancer (NSCLC) samplesconfirming their presentation on primary lung cancer (NSCLC).

TUMAPs identified on multiple lung cancer (NSCLC) and normal tissueswere quantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

Furthermore, the discovery pipeline XPRESI DENT® v2.x allows for thedirect absolute quantitation of MHC-, preferably HLA-restricted, peptidelevels on cancer or other infected tissues. Briefly, the total cellcount was calculated from the total DNA content of the analyzed tissuesample. The total peptide amount for a TUMAP in a tissue sample wasmeasured by nanoLC-MS/MS as the ratio of the natural TUMAP and a knownamount of an isotope-labelled version of the TUMAP, the so-calledinternal standard. The efficiency of TUMAP isolation was determined byspiking peptide:MHC complexes of all selected TUMAPs into the tissuelysate at the earliest possible point of the TUMAP isolation procedureand their detection by nanoLC-MS/MS following completion of the peptideisolation procedure. The total cell count and the amount of totalpeptide were calculated from triplicate measurements per tissue sample.The peptide-specific isolation efficiencies were calculated as anaverage from 10 spike experiments each measured as a triplicate (seeexamples and Table 22).

The present invention provides peptides that are useful in treatingcancers/tumors, preferably lung cancer that over- or exclusively presentthe peptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanlung cancer (NSCLC) samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy lung cells or other normal tissue cells, demonstrating a highdegree of tumor association of the source genes (see example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from lung cancer (NSCLC), but not on normaltissues (see example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. lung cancer cells presenting the derivedpeptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see example 3, example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies, specifically binding fragmentsthereof, antibody-like binders and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Additional methods for the production aredisclosed in WO 2013/057586A1.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral—NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 110, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLRS ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 110,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 110, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 110 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:110 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 110, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 110.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of lung cancer.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 110 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are lung cancer cells or othersolid or hematological tumor cells such as brain cancer, breast cancer,colorectal cancer, esophageal cancer, kidney cancer, liver cancer,ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer,melanoma, merkel cell carcinoma, leukemia (AML, CLL), non-Hodgkinlymphoma (NHL), esophageal cancer including cancer of thegastric-esophageal junction (OSCAR), gallbladder cancer andcholangiocarcinoma (GBC, CCC), urinary bladder cancer (UBC), and uterinecancer (UEC).

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of lung cancer. The present invention also relates to the useof these novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a lung cancer marker(poly)peptide, delivery of a toxin to a lung cancer cell expressing acancer marker gene at an increased level, and/or inhibiting the activityof a lung cancer marker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length lung cancer marker polypeptides or fragmentsthereof may be used to generate the antibodies of the invention. Apolypeptide to be used for generating an antibody of the invention maybe partially or fully purified from a natural source, or may be producedusing recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 110polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the lung cancer marker polypeptideused to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed lung cancers or frozentissue sections. After their initial in vitro characterization,antibodies intended for therapeutic or in vivo diagnostic use are testedaccording to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating lung cancer, theefficacy of the therapeutic antibody can be assessed in various wayswell known to the skilled practitioner. For instance, the size, number,and/or distribution of lung cancer in a subject receiving treatment maybe monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment oflung cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 110, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively eliciting high-or low-avidity antigen-specific T cell responses with high efficiencyfrom blood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 110.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from lung cancer, themedicament of the invention is preferably used to treat lung cancer.

The present invention further relates to a method for producing apersonalized pharmaceutical (composition) for an individual patientcomprising manufacturing a pharmaceutical composition comprising atleast one peptide selected from a warehouse of pre-screened TUMAPs,wherein the at least one peptide used in the pharmaceutical compositionis selected for suitability in the individual patient. In oneembodiment, the pharmaceutical composition is a vaccine. The methodcould also be adapted to produce T cell clones for down-streamapplications, such as TCR isolations, or soluble antibodies, and othertreatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly over-expressed in the tumor tissue of lungcancer patients with various HLA-A HLA-B and HLA-C alleles. It maycontain MHC class I and MHC class II peptides or elongated MHC class Ipeptides. In addition to the tumor associated peptides collected fromseveral lung cancer tissues, the warehouse may contain HLA-A*02 andHLA-A*24 marker peptides. These peptides allow comparison of themagnitude of T-cell immunity induced by TUMAPS in a quantitative mannerand hence allow important conclusion to be drawn on the capacity of thevaccine to elicit anti-tumor responses. Secondly, they function asimportant positive control peptides derived from a “non-self” antigen inthe case that any vaccine-induced T-cell responses to TUMAPs derivedfrom “self” antigens in a patient are not observed. And thirdly, it mayallow conclusions to be drawn, regarding the status of immunocompetenceof the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, lung cancer samples frompatients and blood from healthy donors were analyzed in a stepwiseapproach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (lungcancer) compared with a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromlung cancer patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumine). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from lung cancer cells and since it was determined thatthese peptides are not or at lower levels present in normal tissues,these peptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for lung cancer. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from University Hospital ofHeidelberg; University Hospital of Munich. Normal (healthy) tissues wereobtained from Bio-Options Inc., CA, USA; BioServe, Beltsville, Md., USA;Capital BioScience Inc., Rockville, Md., USA; Geneticist Inc., Glendale,Calif., USA; University Hospital of Geneva; University Hospital ofHeidelberg; Kyoto Prefectural University of Medicine (KPUM); Osaka CityUniversity (OCU); University Hospital Munich; ProteoGenex Inc., CulverCity, Calif., USA; University Hospital of Tubingen.

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, -B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESl source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose lung cancer samples to a baseline of normal tissuesamples.

Presentation profiles of exemplary over-presented peptides are shown inFIG. 1. Presentation scores for exemplary peptides are shown in Table15and Table 16.

TABLE 15 Presentation scores. The table lists HLA-A*02 peptides that are very highly over-presented ontumors compared to a panel of normal tissues(+++), highly over-presented on tumors comparedto a panel of normal (healthy) tissues (++) orover-presented on tumors compared to a panel of normal tissues (+).SEQ ID Peptide Peptide No. Sequence Code Presentation   1 KLLPYIVGVCOL6A3-010 +++   2 FLIPYAIML SLC6A14-001 +++   3 FLYDVVKSL COL6A3-007 ++  4 FVFSFPVSV DUSP4-001 +   5 ALTSTLISV GPNM-002 ++   9 ALSGTLSGVMCM5-001 ++  13 FLFDGSANL COL6A3-008 +++  14 LIQDRVAEV LAMB3-001 +  15ELDRTPPEV SF3B3-001 +  16 LIFDLGGGTFDV HSP-003 +  17 TLLQEQGTKTVKRT-006 +  18 ILLTEQINL PHT-001 +  20 LMTKEISSV PRKDC-001 +  21VLSSGLTAA CSNK2A2-001 +  94 ILVDWLVQV CCNB2-001 +++  96 AMGIAPPKVPRPF3-001 +  97 TLFPVRLLV LPCAT1-001 +  98 VLYPHEPTAV DONSON-001 ++  99ALFQRPPLI DKC-001 + 101 LLLEILHEI ERO-001 + 102 SLLSELQHA GBP5-001 ++103 KLLSDPNYGV TMEM43-001 + 105 IVAESLQQV STA-002 + 111 SLYKGLLSVRAD54B-001 +++ 112 VLAPLFVYL FZD-001 ++ 113 FLLDGSANV COL6A3-002 +++ 114AMSSKFFLV WNT5A-001 ++ 115 YVYQNNIYL FAP-003 + 116 KIQEMQHFL MMP12-003+++ 117 ILIDWLVQV CCNB1-002 ++ 118 SLHFLILYV ATP-001 + 119 IVDDITYNVFN1-001 ++ 120 KIQEILTQV IGF2BP3-001 +++ 121 RLLDSVSRL LAMC2-001 + 122KLSWDLIYL CERC-001 + 123 GLTDNIHLV MXRA5-002 ++ 124 NLLDLDYEL COL6A3-003+++ 125 RLDDLKMTV LAMC2-002 ++ 126 KLLTEVHAA ADAM8-001 ++ 127 ILFPDIIARAMAGEF1-001 + 128 TLSSIKVEV MXRA5-001 +++ 129 GLIEIISNA SNRNP20-001 + 130KILEDVVGV TPX2-001 + 131 ALVQDLAKA CCNB1-001 + 132 ALFVRLLALA TGFBI-001++ 133 RLASYLDKV KRT-007 + 134 TLVVYRAPEV CDK4-001 + 136 ALVDHTPYLVCAN-002 + 137 FLVDGSWSV COL12A1-002 ++ 138 ALNEEAGRLLL UBE2S-001 ++ 139SLIEDLILL SMYD3-001 ++ 142 VLLPVEVATHYL SLC34A2-001 + 143 AIVDKVPSVCOPG1-001 + 144 KIFDEILVNA TOP-001 + 145 AMTQLLAGV TNC-001 + 146FQYDHEAFL RCN1-001 ++ 148 ALFGALFLA PLT-001 + 149 KLVEFDFLGA TACC3-001 +150 GVLENIFGV PCNXL3-001 + 152 ILQDRLNQV CDC6-001 + 153 ALYDSVILLDIO2-001 ++ 156 TVAEVIQSV KIF26B-001 +

TABLE 16 Presentation scores. The table lists HLA-A*24peptides that are very highly over-presented ontumors compared to a panel of normal tissues(+++), highly over-presented on tumors comparedto a panel of normal tissues (++) or over-presented on tumors compared to a panel of normal tissues (+). SEQ IDPeptide No. Sequence Peptide Code Presentation  23 VYTSWQIPQKF CCL18-001+++  24 NYPKSIHSF MMP12-005 +++  25 RFMDGHITF LAMP3-001 +++  26RYLEKFYGL MMP12-006 +++  27 RYPPPVREF COL6A3-012 +++  28 RYLDSLKAIVFCENPN-001 +++  29 YYTKGFALLNF PLOD2-002 +++  30 KYLEKYYNL MMP1-001 +++ 31 SYLDKVRAL KRT-008 +++  32 EYQPEMLEKF COL6A3-013 +++  33 TYSEKTTLFMUC16-001 +++  34 VFMKDGFFYF MMP1-002 +++  35 TYNPEIYVI ITGA2-002 +++ 36 YYGNTLVEF OLFML2B-001 +++  37 RYLEYFEKI TTC13-001 +++  38VFLNRAKAVFF GPNM-003 +++  39 KFLEHTNFEF DOCK2-001 +++  40 IYNPSMGVSVLPVRL1-001 +++  41 TYIGQGYII FKBP10-002 +++  42 VYVTIDENNIL ABCC1-001 +++ 43 RYTLHINTL ALOX15B-001 +++  44 IYNQIAELW SMPDL3B-001 +++  45KFLESKGYEF GFPT2-002 +++  46 NYTNGSFGSNF DDX5-007 +++  47 RYISPDQLADLENO1-001 +++  48 YYYGNTLVEF OLFML2B-002 +++  49 QYLFPSFETF KLRD-001 +++ 50 LYIGWDKHYGF PSMA4-001 +++  51 NYLLESPHRF PLE-001 +++  52 SYMEVPTYLNFLAPTM5-001 +++  53 IYAGQWNDF COLEC12-001 +++  54 AYKDKDISFF ZBED5-001+++  55 IYPVKYTQTF PERP-001 +++  56 RYFPTQALNF SLC-003 +++  57 SYSIGIANFCOL12A1-003 +++  58 VYFKPSLTPSGEF NUP153-001 +++  59 HYFNTPFQL PPTC-001+++  60 SYPAKLSFI FLJ44796-001 +++  61 RYGSPINTF C6orf132-001 +++  62AYKPGALTF AIFM2-001 +++  63 LYINKANIW G2E-002 +++  64 VYPLALYGF XPR-001+++  65 IYQRWKDLL SAMD9L-001 +++  66 DYIPQLAKF GLS-001 +++  67IFLDYEAGHLSF TRIM11-001 +++  68 RYLFVVDRL MREG-001 +++  69 TYAALNSKATFIQGAP1-001 +++  70 VYHSYLTIF TRPM2-001 +++  71 TYLTNHLRL ZNF697-001 +++ 72 YYVDKLFNTI RASA2-001 +++  73 RYLHVEGGNF RBPJ-001 +++  74 EYLPEFLHTFABCA13-003 +++  75 AYPDLNEIYRSF SP14-001 +++  76 VYTZIQSRF DYR-001 +++ 77 RYLEAGAAGLRW HSPBP-001 +++  78 IYTRVTYYL TPS-001 +++  79 RYGGSFAELKDM6B-001 ++  81 KYIEAIQWI DCSTA-001 +++  82 FYQGIVQQF TUBGCP3-001 +++ 83 EYSDVLAKLAF AHD-001 +++  84 TFDVAPSRLDF NOM-001 +++  85 PFLQASPHFFAM83A-001 +++ 159 TYKYVDINTF MMP12-004 +++ 160 SYLQAANAL COL6A3-001 +++161 LYQILQGIVF CDC2-001 +++

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalization, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0. Exemplary expression profiles of source genes of the presentinvention that are highly over-expressed or exclusively expressed inlung cancer are shown in FIG. 2. Expression scores for further exemplarygenes are shown in Table 17 and Table 18.

TABLE 17 Expression scores. The table lists HLA-A*02peptides from genes that are very highly over-expressed in tumors compared to a panel ofnormal tissues (+++), highly over-expressed intumors compared to a panel of normal tissues(++) or over-expressed in tumors compared toa panel of normal tissues (+). SEQ ID Peptide Gene No. Sequence CodeExpression   2 FLIPYAIML SLC6A14-001 ++   5 ALTSTLISV GPNM-002 +   6SLQGSIMTV SFT-001 ++   8 ALLNILSEV UBA6-002 ++  11 YLNVQVKEL SMC4-002 ++ 12 IVDRTTTVV SLC1A4-001 +  14 LIQDRVAEV LAMB3-001 ++  16 LIFDLGGGTFDVHSP-003 +  18 ILLTEQINL PHT-001 +  19 VLTSDSPAL GPNM-001 +  95 KIIGIMEEVMSH6-001 ++  97 TLFPVRLLV LPCAT1-001 + 105 IVAESLQQV STA-002 ++ 106SILEHQIQV MCM4-001 ++ 108 TLLDFINAV UBA6-001 ++ 115 YVYQNNIYL FAP-003 +116 KIQEMQHFL MMP12-003 +++ 117 ILIDWLVQV CCNB1-002 + 119 IVDDITYNVFN1-001 + 120 KIQEILTQV IGF2BP3-001 ++ 121 RLLDSVSRL LAMC2-001 ++ 125RLDDLKMTV LAMC2-002 ++ 130 KILEDVVGV TPX2-001 + 131 ALVQDLAKACCNB1-001 + 133 RLASYLDKV KRT-007 + 136 ALVDHTPYL VCAN-002 + 140TLYPHTSQV VCAN-001 + 141 NLIEKSIYL DST-001 + 142 VLLPVEVATHYLSLC34A2-001 ++ 143 AIVDKVPSV COPG1-001 + 144 KIFDEILVNA TOP-001 ++ 152ILQDRLNQV CDC6-001 + 158 KLDETNNTL DST-002 +

TABLE 18 Expression scores. The table lists HLA-A*24peptides from genes that are very highly over-expressed in tumors compared to a panel of normaltissues (+++), highly over-expressed in tumorscompared to a panel of normal tissues (++) orover-expressed in tumors compared to a panel of normal tissues (+).SEQ ID Peptide Gene No. Sequence Code Expression  23 VYTSWQIPQKFCCL18-001 +++  24 NYPKSIHSF MMP12-005 +++  25 RFMDGHITF LAMP3-001 +++ 26 RYLEKFYGL MMP12-006 +++  28 RYLDSLKAIVF CENPN-001 ++  29 YYTKGFALLNFPLOD2-002 +  30 KYLEKYYNL MMP1-001 +  31 SYLDKVRAL KRT-008 +  34VFMKDGFFYF MMP1-002 +  35 TYNPEIYVI ITGA2-002 +  38 VFLNRAKAVFFGPNM-003 +  39 KFLEHTNFEF DOCK2-001 +  43 RYTLHINTL ALOX15B-001 +  47RYISPDQLADL ENO1-001 + 159 TYKYVDINTF MMP12-004 +++ 161 LYQILQGIVFCDC2-001 +

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for 84 HLA-A*02 restricted TUMAPs ofthe invention so far, demonstrating that these peptides are T-cellepitopes against which CD8+ precursor T cells exist in humans (Table19).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using

CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthydonors obtained from the University clinics Mannheim, Germany, afterinformed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nurnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 163) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.164), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRI I SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Lung Cancer Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 3peptides of the invention are shown in FIGS. 3A-3E together withcorresponding negative controls. Results for 84 peptides from theinvention are summarized in Table 19.

TABLE 19 in vitro immunogenicity of HLA-A*02 peptides of the inventionExemplary results of in vitro immunogenicity experiments conducted bythe applicant for the peptides of the invention. Seq ID Peptide codewells donors 1 COL6A3-010 ++ ++++ 2 SLC6A14-001 + ++++ 3 COL6A3-007 +++++ 4 DUSP4-001 ++ ++++ 5 GPNM-002 + ++++ 6 SFT-001 ++ ++++ 7KRT80-001 + ++ 8 UBA6-002 ++ ++++ 9 MCM5-001 + ++++ 10 KRT15-001 ++++++++ 11 SMC4-002 + ++++ 12 SLC1A4-001 ++++ ++++ 13 COL6A3-008 + ++ 14LAMB3-001 + ++++ 15 SF3B3-001 ++ ++++ 16 HSP-003 + +++ 17 KRT-006 + ++18 PHT-001 + ++ 19 GPNM-001 + ++++ 21 CSNK2A2-001 + + 22 PTPN13-001 + +94 CCNB2-001 ++ ++++ 95 MSH6-001 ++++ ++++ 96 PRPF3-001 ++++ ++++ 97LPCAT1-001 +++ ++++ 98 DONSON-001 + ++++ 99 DKC-001 ++ ++++ 100BUB1B-001 ++ ++++ 101 ERO-001 + +++ 102 GBPS-001 + ++ 103 TMEM43-001 +++++ 104 COG4-001 + ++++ 105 STA-002 + + 106 MCM4-001 + + 107PSMD14-002 + +++ 108 UBA6-001 + +++ 109 CCZ-001 + +++ 111 RAD54B-001 ++++++ 112 FZD-001 +++ ++++ 113 COL6A3-002 ++ ++++ 114 WNT5A-001 ++ ++++115 FAP-003 + ++++ 116 MMP12-003 + ++++ 117 CCNB1-002 ++ ++++ 118ATP-001 ++++ ++++ 119 FN1-001 ++ ++++ 120 IGF2BP3-001 + ++++ 121LAMC2-001 ++ ++++ 122 CERC-001 +++ ++++ 123 MXRA5-002 + ++++ 124COL6A3-003 + ++++ 125 LAMC2-002 + ++++ 126 ADAM8-001 + ++++ 127MAGEF1-001 +++ ++++ 128 MXRA5-001 + ++++ 129 SNRNP20-001 ++ ++++ 130TPX2-001 ++ ++++ 131 CCNB1-001 ++ ++++ 132 TGFBI-001 ++ ++++ 133 KRT-007++ ++++ 134 CDK4-001 ++++ ++++ 135 GFPT2-001 +++ ++++ 136 VCAN-002 +++++ 137 COL12A1-002 + ++ 138 UBE2S-001 + ++++ 139 SMYD3-001 + ++ 140VCAN-001 ++ ++++ 141 DST-001 + ++++ 142 SLC34A2-001 + ++ 143 COPG1-001 ++++ 144 TOP-001 + ++ 145 TNC-001 + ++ 147 BNC1-001 + ++++ 148 PLT-001 +++ 149 TACC3-001 + +++ 150 PCNXL3-001 + +++ 151 DROSHA-001 + ++++ 152CDC6-001 + +++ 153 DIO2-001 + + 154 ABCA13-001 + ++++ 155 ABCA13-002 ++++ 156 KIF26B-001 + + 157 SERPINB3-001 + +++ 158 DST-002 + ++ <20% = +,20%-49% = ++, 50%-69% = +++, >=70% = ++++

TABLE 20 in vitro immunogenicity of HLA-A*24 peptides of the inventionExemplary results of in vitro immunogenicity experiments conducted bythe applicant for the peptides of the invention. Seq ID Peptide codewells donors 23 CCL18-001 + + 25 LAMP3-001 + ++ 26 MMP12-006 + ++++ 27COL6A3-012 ++ ++++ 28 CENPN-001 + ++++ 29 PLOD2-002 + +++ 30 MMP1-001 ++++++ 32 COL6A3-013 + + 33 MUC16-001 + ++ 34 MMP1-002 + ++ 35 ITGA2-002++ ++++ 36 OLFML2B-001 ++ ++++ 37 TTC13-001 +++ ++++ 39 DOCK2-001 + ++40 PVRL1-001 + ++ 41 FKBP10-002 + ++++ 42 ABCC1-001 + ++ 43ALOX15B-001 + ++ 44 SMPDL3B-001 ++ ++++ 46 DDX5-007 + + 47 ENO1-001 ++++ 48 OLFML2B-002 + ++ 49 KLRD-001 + ++++ 50 PSMA4-001 + ++++ 52LAPTM5-001 + ++ 53 COLEC12-001 + +++ 54 ZBED5-001 + ++++ 56 SLC-003 + +57 COL12A1-003 + ++++ 58 NUP153-001 + + 59 PPTC-001 ++ ++++ 61C6orf132-001 + +++ 62 AIFM2-001 + ++ 63 G2E-002 + ++ 64 XPR-001 ++ ++++65 SAMD9L-001 + +++ 66 GLS-001 + ++++ 67 TRIM11-001 + + 68 MREG-001 ++++++++ 69 IQGAP1-001 + + 70 TRPM2-001 ++ ++++ 71 ZNF697-001 ++ ++++ 72RASA2-001 + ++ 73 RBPJ-001 + ++ 74 ABCA13-003 + ++ 75 SP14-001 + + 76DYR-001 + ++ 77 HSPBP-001 + + 78 TPS-001 ++ ++++ 79 KDM6B-001 + ++ 81DCSTA-001 ++ ++++ 82 TUBGCP3-001 + ++ 83 AHD-001 + +++ 85 FAM83A-001 ++++++ 159 MMP12-004 + +++ 160 COL6A3-001 + ++ 161 CDC2-001 + ++++ <20% =+, 20%-49% = ++, 50%-69% = +++, >=70% = ++++

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >85%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other pharmaceutically acceptable salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1h at37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1h at 37° C., washed again and detected with TMB solutionthat is stopped with NH2SO4. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 21 MHC class I binding scores Peptide Seq. ID Peptide codeexchange 31 KRT-008 +++ 45 GFPT2-002 +++ 51 PLE-001 +++ 55 PERP-001 +++60 FLJ44796-001 +++ 80 FXR1-001 +++ <20% = +, 20%-49% = ++, 50%-75% =+++, >=75% = ++++

Example 6

Absolute Quantitation of Tumor Associated Peptides Presented on the CellSurface

The generation of binders, such as antibodies and/or TCRs, is alaborious process, which may be conducted only for a number of selectedtargets. In the case of tumor-associated and—specific peptides,selection criteria include but are not restricted to exclusiveness ofpresentation and the density of peptide presented on the cell surface.In addition to the isolation and relative quantitation of peptides asdescribed in example 1, the inventors did analyze absolute peptidecopies per cell. The quantitation of TUMAP copies per cell in solidtumor samples requires the absolute quantitation of the isolated TUMAP,the efficiency of TUMAP isolation, and the cell count of the tissuesample analyzed. Experimental steps are described below.

Peptide Quantitation by Nano LC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, acalibration curve was generated for each peptide using the internalstandard method. The internal standard is a double-isotope-labelledvariant of each peptide, i.e. two isotope-labelled amino acids wereincluded in TUMAP synthesis. It differs from the tumor-associatedpeptide only in its mass but shows no difference in otherphysicochemical properties (Anderson et al., 2012). The internalstandard was spiked to each MS sample and all MS signals were normalizedto the MS signal of the internal standard to level out potentialtechnical variances between MS experiments. The calibration curves wereprepared in at least three different matrices, i.e. HLA peptide eluatesfrom natural samples similar to the routine MS samples, and eachpreparation was measured in duplicate MS runs. For evaluation, MSsignals were normalized to the signal of the internal standard and acalibration curve was calculated by logistic regression. For thequantitation of tumor-associated peptides from tissue samples, therespective samples were also spiked with the internal standard, the MSsignals were normalized to the internal standard and quantified usingthe peptide calibration curve.

Efficiency of Peptide/MHC Isolation

As for any protein purification process, the isolation of proteins fromtissue samples is associated with a certain loss of the protein ofinterest. To determine the efficiency of TUMAP isolation, peptide/MHCcomplexes were generated for all TUMAPs selected for absolutequantitation. To be able to discriminate the spiked from the naturalpeptide/MHC complexes, single-isotope-labelled versions of the TUMAPswere used, i.e. one isotope-labelled amino acid was included in TUMAPsynthesis. These complexes were spiked into the freshly prepared tissuelysates, i.e. at the earliest possible point of the TUMAP isolationprocedure, and then captured like the natural peptide/MHC complexes inthe following affinity purification. Measuring the recovery of thesingle-labelled TUMAPs therefore allows conclusions regarding theefficiency of isolation of individual natural TUMAPs. The efficiency ofisolation was analyzed in a low number of samples and was comparableamong these tissue samples. In contrast, the isolation efficiencydiffers between individual peptides. This suggests that the isolationefficiency, although determined in only a limited number of tissuesamples, may be extrapolated to any other tissue preparation. However,it is necessary to analyze each TUMAP individually as the isolationefficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue

In order to determine the cell count of the tissue samples subjected toabsolute peptide quantitation, the inventors applied DNA contentanalysis. This method is applicable to a wide range of samples ofdifferent origin and, most importantly, frozen samples (Forsey andChaudhuri, 2009; Alcoser et al., 2011; Silva et al., 2013). During thepeptide isolation protocol, a tissue sample is processed to a homogenouslysate, from which a small lysate aliquot is taken. The aliquot isdivided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit,Qiagen, Hilden, Germany). The total DNA content from each DNA isolationis quantified using a fluorescence-based DNA quantitation assay (QubitdsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at leasttwo replicates. In order to calculate the cell number, a DNA standardcurve from aliquots of single healthy blood cells, with a range ofdefined cell numbers, was generated. The standard curve is used tocalculate the total cell content from the total DNA content from eachDNA isolation. The mean total cell count of the tissue sample used forpeptide isolation is extrapolated considering the known volume of thelysate aliquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculatedthe number of TUMAP copies per cell by dividing the total peptide amountby the total cell count of the sample, followed by division throughisolation efficiency. Copy cell numbers for selected peptides are shownin Table 22.

TABLE 22 Absolute copy numbers. The table lists the results of absolutepeptide quantitation in NSCLC tumor samples. SEQ ID Copies per Number ofNo. Peptide Code cell (median) samples 13 COL6A3-008 +++ 18 23 CCL18-001+++ 19 24 MMP12-005 +++ 11 25 LAMP3-001 ++++ 7 26 MMP12-006 +++ 17 27COL6A3-012 +++ 12 29 PLOD2-002 ++ 22 30 MMP1-001 +++ 11 32 COL6A3-013 ++20 33 MUC16-001 ++ 22 35 ITGA2-002 ++ 19 36 OLFML2B-001 +++ 22 37TTC13-001 +++ 13 38 GPNM-003 +++ 5 41 FKBP10-002 ++ 19 98 DONSON-001 +18 100 BUB1B-001 + 11 111 RAD54B-001 + 16 113 COL6A3-002 ++ 19 114WNT5A-001 ++ 17 115 FAP-003 ++ 17 120 IGF2BP3-001 ++ 14 121 LAMC2-001 ++10 123 MXRA5-002 + 17 124 COL6A3-003 +++ 18 126 ADAM8-001 + 15 128MXRA5-001 ++ 19 137 COL12A1-002 ++ 12 140 VCAN-001 + 19 141 DST-001 ++12 147 BNC1-001 + 10 158 DST-002 ++ 7 159 MMP12-004 ++ 22 160 COL6A3-001++++ 23 161 CDC2-001 +++ 18 The median numbers of copies per cell areindicated for each peptide: <100 = +, >=100 = ++, >=1,000 +++, >=10,000= ++++. The number of samples, in which evaluable, high quality MS dataare available is indicated.

Example 6

HLA Class II T-Cell Proliferation Assays

The experiments as follows summarize the results of T-cell ProliferationAssays of selected MHC class II TUMAPs. Nine of 10 tested peptideantigens tested positive for immunogenicity. Eleven out of 21 evaluableT-cell samples showed a positive response for at least one peptide.Individual peptide antigens stimulated the CD4+ T-cell proliferation inup to 6 donors. These numbers are comparable to the results for fivereference peptides tested in the same assay runs and with immunogenicitydemonstrated for the majority of patients in clinical vaccine trialsettings. Thus, it can be concluded that the newly tested peptides alsohave a high potential to induce T-cell responses in vaccine trials.

In order to characterize selected peptides for their potentialespecially as vaccine candidates, their in vitro immunogenicity wasdetermined by analysis of T-cell proliferation using a commercial T-cellProliferation Assay from the company ProImmune.

Healthy donor CD8-depleted blood cells samples were tested with theselected peptides. The peptides that induce the proliferation of CD4+ Tcells can potentially result in the development of a helper T-cellimmune response, and therefore are considered to be immunogenic. Theproliferation of CD4+ T cells was determined using carboxyfluoresceinsuccinimidyl ester (CFSE) labelling. In proliferating cells, CFSE isdistributed evenly to dividing cells. Thus, the proliferation can bemeasured as a decrease in CFSE fluorescence in the cells as analyzed.

TABLE 23 Selected HLA class II peptides. Sequence ID NO: Peptide IDSequence Origin  86 MMP12-007 LSADDIRGIQSLYGDPK This app.  87COL11A1-001 EGDIQQFLITGDPKAAYDY This app.  92 COL1A2-001NKPSRLPFLDIAPLDIGGAD This app.  91 COL5A2-001 VARLPIIDLAPVDVGGTDThis app.  93 FN1-002 SRPQAPITGYRIVYSPSV This app.  88 ITGB6-001NPVSQVEILKNKPLSVG This app.  90 LAMC2-003 DAVQMVITEAQKVDTR This app. 110LAMP3-002 IQLIVQDKESVFSPR This app.  89 IGF2BP3-002 KLYIGNLSENAAPSThis app. 162 POSTN-002 TNGVIHVVDKLLYPADT This app. 165 BIR-002TLGEFLKLDRERAKN Pos. contr. 166 MET-005 TFSYVDPVITSISPKYG Pos. contr.167 MMP-001 SQDDIKGIQKLYGKRS Pos. contr. 168 CEA-006 SPQYSWRINGIPQQHTPos. contr. 169 TGFBI-004 TPPIDAHTRNLLRNH Pos. contr.

Principle of Test

Peripheral blood mononuclear cell (PBMC) samples from healthy humandonors were selected from the ProImmune cell bank based on HLA-DRB1allele expression. CD8+ T-cells were depleted from donor blood samplesprior to use to avoid a false-positive response. The remaining CD4+ Tcells were labelled with CFSE and subsequently incubated with 5 μM ofeach selected peptide. Each peptide was tested in six replicated wells.The background was measured on each plate in six unstimulated controlwells.

After an incubation period of 7 days the cells were co-stained withanti-CD4 antibody and analyzed by flow cytometry. The degree ofproliferation was determined by measuring a reduction in CFSE intensity.

The evaluation of flow cytometric data was performed using FlowJoSoftware (Tree Star, Inc.). The results of flow cytometric analysis wereexpressed as the ratio of the CD4⁺ dim population to the total CD4⁺population. The degree of proliferation was expressed as percentage ofstimulation above background, i.e. proportion of antigen stimulated CD4⁺CFSE dim cells minus the proportion of CD4⁺ CFSE dim cells fromunstimulated control wells. For each sample, a mean and thecorresponding standard error of the mean (SEM) of the six replicateswere calculated.

Selection of Donors

Donors were chosen by HLA-DRB1 allele expression. The other two HLAclass II loci (DQ and DP) have not been included into the analysis. Theinteresting DRB1 alleles were selected according to the frequencies ofpredicted peptide binding based on SYFPEITHI algorithm (Rammensee etal., 1999). For HLA-DR, binding was defined by a SYFPEITHI bindingprediction score equal or greater than 18. This threshold score forbinding was defined based on the analysis of binding scores of knownpublished promiscuous HLA-DR ligands (Table 24).

TABLE 24 SYFPEITHI prediction scores for HLA-DR binding of peptides thathave been shown experimentally to bind to several HLA-DRalleles. Prediction scores are only shown, if binding to theindicated DR allele has been shown experimentally. If highresolution information of DR alleles is not available this ismarked by*. 23 of 26 (89%) SYFPEITHI scores are > = 18, if thebinding was experimentally shown for an allele. DRB1* allele Peptide0101 0301 0401 0701 1101 1501 SSX2₄₅₋₅₉ — — — 18 24 — KIFYVYMKRKYEAMTSEQ ID NO: 170 MAGE A3₁₁₁₋₁₂₅ 24* — 26* — 23* — RKVAELVHFLLLKYRSEQ ID NO: 171 MAGE A3₁₄₆₋₁₆₀ 31* — 28* 24* 24* — FFPVIFSKASSSLQLSEQ ID NO: 172 MAGE A3₁₉₁₋₂₀₅ 24* — 20* — 14* — GDNQIMPKAGLLIIVSEQ ID NO: 173 MAGE A3₂₈₁₋₃₉₅ 27* — 28* — 28* — TSYVKVLHHMVKISGSEQ ID NO: 174 NY-ESO-1₁₂₁₋₁₃₈ 22 — — 24 14 — VLLKEFTVSGNILTIRLTSEQ ID NO: 175 HER2/neU₈₈₃₋₈₉₉ 25 — 22 — — — KVPIKWMALESILRRRFSEQ ID NO: 176 PADRE 26 11 28 28 17 24 [D-Ala]K[L-cyclohexyl-Ala]VAAWTLKAA[D-Ala] SEQ ID NO: 177

All DRB1 alleles with binding frequency over 20% over all selectedpeptides were requested to be included into the donor panel) byProImmune. 4 other rare DRB1 alleles (DRB1*10:01, DRB1*16:01,DRB1*08:01, and DRB1*13:03) were requested additionally. The assembleddonor panel is shown in Table 26.

TABLE 25 Binding capacity of selected peptides to various HLA-DRB1alleles with known binding motif: A SYFPEITHI score over 17 was countedwith 1 as a binding event. The last column shows the percentage ofbinding events over all selected peptides. MMP12- COL11A1- COL1A2-COL5A2- FN1- ITGB6- LAMC2- LAMP3- IGF2BP3- MHC 007 001 001 001 002 001003 002 002 DRB1*0401 1 1 1 1 1 1 1 1 1 DRB1*0404 1 1 1 1 1 1 1 1 1DRB1*0101 1 1 1 1 1 1 1 1 1 DRB1*0301 1 1 1 1 0 1 1 1 1 DRB1*1104 1 1 11 1 1 1 1 1 DRB1*0405 1 1 1 1 1 1 1 1 1 DRB1*0402 1 1 1 1 0 1 1 1 1DRB1*0701 1 1 1 1 1 1 1 1 1 DRB1*1501 1 1 1 1 1 1 1 1 0 DRB1*1301 0 1 11 1 0 1 0 1 DRB1*1502 1 1 1 1 1 1 0 0 1 DRB1*1101 1 0 1 0 0 1 0 0 1DRB1*0901 0 0 0 1 1 1 0 0 0 DRB1*1302 0 1 0 0 0 0 0 0 0 DRB1*0802 0 1 00 0 0 0 0 0 DRB1*0803 0 0 0 0 0 0 0 0 0 % of POSTN- BIR- MET- MMP- CEA-TGFBI- Peptide MHC 002 002 005 001 006 004 binders DRB1*0401 1 1 1 1 1 1100% DRB1*0404 1 1 1 1 1 1 100% DRB1*0101 1 1 1 1 1 0  93% DRB1*0301 1 11 1 1 1  93% DRB1*1104 1 1 1 1 0 1  93% DRB1*0405 1 1 1 1 1 0  93%DRB1*0402 1 1 1 1 1 0  87% DRB1*0701 1 0 1 0 0 1  80% DRB1*1501 0 1 1 10 0  73% DRB1*1301 1 1 1 1 0 0  67% DRB1*1502 0 1 1 1 0 0  67% DRB1*11011 1 1 1 0 0  53% DRB1*0901 1 0 1 0 1 0  40% DRB1*1302 1 1 0 0 0 0  20%DRB1*0802 1 1 0 0 0 0  20% DRB1*0803 1 1 0 0 0 0  13%

TABLE 26 Donor panel. HLA-DRB1 allele distribution of 21 selected donorsDonor ID DRB1_1 DRB1_2 D778 *04:04 *10:01 D780 *01:01 *04:01 D789 *13:01*16:01 D799 *03:01 *09:01 D800 *15:02 *16:01 D801 *11:01 *15:01 D813*07:01 *15:01 D816 *01:01 *04:05 D817 *10:01 *13:01 D820 *01:01 *07:01D822 *04:04 *08:01 D829 *07:01 *11:01 D836 *13:01 *13:03 D845 *03:01*11:04 D857 *03:01 *04:05 D906 *15:01 *15:02 D940 *04:01 *15:01 D946*11:01 *14:01 D951 *03:01 *04:04 D962 *03:01 *09:01 D973 *03:01 *11:04

Results of In Vitro Immunogenicity

The antigen-stimulated proliferation of CD4+ T cells was considered asan indicator of in vitro immunogenicity and was investigated in acommercially available T-cell Proliferation Assay from ProImmune. Thedegree of antigen-stimulated CD4+ T-cell proliferation was expressed asa percentage of stimulation above background. A response over 0.02%stimulation above background with SEM=2 (i.e. values two standard errorsgreater than background) was considered to be positive.

Nine of 10 selected peptide antigens (with the exception of FN1-002)were tested positive. Eleven out of 21 evaluable T-cell samples showed apositive response for at least one peptide (FIG. 4). Individual peptideantigens stimulated the CD4+ T-cell proliferation in up to 6 donors.

Comparison of In Vivo to In Vitro Immunogenicity

The T-cell proliferation analysis included 5 peptides with known in vivoimmunogenicity as positive controls. The in vivo immunogenicity of thesepeptides was determined in blood samples of patients vaccinated withthese peptides in clinical trials using intracellular cytokine staining(ICS) of CD4 T cells.

In principle, ICS assays analyze the quality of specific T cells interms of effector functions. Therefore the peripheral mononuclear cells(PBMCs) were re-stimulated in vitro with the peptide of interest, areference peptide and a negative control (here MOCK). Following there-stimulated cells were stained for IFN-gamma, TNF-alpha, IL-2 andIL-10 production, as well as expression of the co-stimulatory moleculeCD154. The counting of stained cells was performed on a flow cytometer(FIG. 5).

The immunogenicity analysis revealed 100% immune response by vaccinationwith IMA950 peptides (BIR-002 and MET-005) in 16 patients (studyIMA950-101) and 44% to 86% immune response by vaccinaton with IMA910peptides (CEA-006, TGFBI-004 and MMP-001) in 71 patients (studyIMA910-101) (FIG. 6).

The results of in vitro immunogenicity of peptides with known in vivoimmunogenicity were compared to the selected peptides (Table 27). Theanalysis showed that the positive control peptides stimulated a CD4+T-cell proliferation in 7 of 21 investigated donor samples. The strengthof stimulation response on average ranged from 0.09 to 0.31% above thebackground in up to 4 donor samples per peptide. For example, thestrenght of stimulation for BIR-002 was 0.24%. BIR-002 was found to behighly immunogenic in different clinical trials. BIR-002 was tested as acomponent of a prostate cancer-specific peptide vaccine in a clinicaltrial with 19 evaluable patients expressing different HLA-DR alleles(Feyerabend et al., 2009). Sixteen (84%) patients mounted a strong CD4+T-cell response against BIR-002 (Widenmeyer et al., 2008) demonstratingits high immunogenicity potential. In the IMA950 trial, 100% (n=16) ofthe patients showed an immune response against BIR-002.

By comparison, with exception of FN1-002, the selected peptides for thecurrent analysis stimulated the CD4+ T-cell proliferation in overall 11investigated donor samples. Thereby, the strength of stimulationresponse in average ranged from 0.19 to 0.48% above the background in upto 6 donors per peptide. These values were similar to the strength ofstimulation response of the highly immunogenic peptide BIR-002.Interestingly, for all positive control peptides the fraction ofpositive donor samples in the in vitro immunogenicity assay (range:4-19%) was considerably lower than the fraction of patients mounting animmune response against these peptides in clinical trials (range:44-100%). This observation indicates that the current in vitroimmunogenicity assay setup is rather conservative and is likely tounderestimate immunogenicity of the peptides in a clinical setting.Thus, it can be expected that 9 of the 10 investigated peptides arehighly likely to induce an in vivo immune response in clinical trials inthe majority of patients.

TABLE 27 Results of T-cell proliferation assay of selected peptides andpositive control peptides with known in vivo immunogenicity. Number ofStrength of response: Sequence positive mean in % above the ID NoPeptide ID donors background 86 MMP12-007 2 0.28 87 COL11A1-001 5 0.1992 COL1A2-001 2 0.48 91 COL5A2-001 2 0.21 88 ITGB6-001 6 0.23 90LAMC2-003 3 0.27 110 LAMP3-002 5 0.25 89 IGF2BP3-002 3 0.40 162POSTN-002 2 0.39 165 BIR-002 3 0.24 166 MET-005 4 0.31 167 MMP-001 20.09 168 CEA-006 1 0.20 169 TGFBI-004 2 0.19

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1. A method of eliciting an immune response in a patient who has canceroverexpressing a MUC16 polypeptide comprising SEQ ID NO: 33, comprisingadministering to said patient a population of activated T cells thatkill cancer cells, wherein the activated T cells are cytotoxic CD8+ Tcells produced by contacting T cells with an antigen presenting cellthat presents a peptide consisting of the amino acid sequence of SEQ IDNO: 33 in a complex with an MHC class I molecule on the surface of theantigen presenting cell in vitro, for a period of time sufficient toactivate said T cell, wherein the cancer is lung cancer or gastriccancer.
 2. The method of claim 1, wherein the T cells are autologous tothe patient.
 3. The method of claim 1, wherein the T cells are obtainedfrom a healthy donor.
 4. The method of claim 1, wherein the T cells areobtained from tumor infiltrating lymphocytes or peripheral bloodmononuclear cells.
 5. The method of claim 1, wherein the activated Tcells are expanded in vitro.
 6. The method of claim 1, wherein theantigen presenting cell is infected with a recombinant virus expressingthe peptide.
 7. The method of claim 6, wherein the antigen presentingcell is a dendritic cell or a macrophage.
 8. The method of claim 5,wherein the expansion is in the presence of an anti-CD28 antibody andIL-12.
 9. The method of claim 1, wherein the population of activated Tcells are administered in the form of a composition.
 10. The method ofclaim 9, wherein the composition further comprises an adjuvant.
 11. Themethod of claim 10, wherein the adjuvant is selected from the groupconsisting of anti-CD40 antibody, imiquimod, resiquimod, GM-CSF,cyclophosphamide, Sunitinib, bevacizumab, interferon-alpha,interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) andderivatives, RNA, sildenafil, and particulate formations withpoly(lactide coglycolide) (PLG), virosomes, interleukin (IL)-1, IL-2,IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 12. The method ofclaim 1, wherein the MEW molecule is HLA-A*24.
 13. The method of claim1, wherein the immune response is cytotoxic T cell response.
 14. Themethod of claim 11, wherein the adjuvant comprises IL-2.
 15. The methodof claim 11, wherein the adjuvant comprises IL-7.
 16. The method ofclaim 11, wherein the adjuvant comprises IL-12.
 17. The method of claim11, wherein the adjuvant comprises IL-15.
 18. The method of claim 11,wherein the adjuvant comprises IL-21.
 19. The method of claim 1, whereinthe cancer is lung cancer.
 20. The method of claim 1, wherein the canceris gastric cancer.